Crosslinked hydrogels with controlled permeability

Abstract

There is provided a method of printing a hydrogel patch containing live cells. A mixture comprising alginate, a first polymer, a second polymer, and live cells are extruded through a needle or nozzle. The first polymer is functionalized with a homocysteine thiolactone and the second polymer is functionalized with a pendant vinyl group. While extruding, the needle or the nozzle to form a precursor of the hydrogel patch. The alginate is gelled, preferably by submersion of the precursor of the hydrogel in a bath containing calcium, strontium, barium, iron, or zinc ions to gel the alginate. The first polymer crosslinks with the pendant vinyl group on the second polymer to obtain the hydrogel patch containing live cells.

Description

CROSSLINKED HYDROGELS WITH CONTROLLED PERMEABILITYCROSS-REFERENCE TO A RELATED APPLICATION

[0001] This patent application claims priority from U.S. provisional application number 63 / 662,627 filed June 21 , 2024, which is incorporated herein by reference in its entirety.TECHNICAL FIELD

[0002] This disclosure relates to the field of hydrogels, particularly semi-permeable hydrogels that are useful in cellular applications, such as cell encapsulation.BACKGROUND OF THE ART

[0003] Hydrogels have long been recognized as useful materials to interface with cells and tissues as it is recognized that hydrogels can mimic certain properties of natural tissues, and can hence be thought of as synthetic extracellular matrix (ECM) materials. Examples range from synthetic crosslinked hydrogels based on hydroxyethyl methacrylate (HEMA) used as contact lenses, to Matrigel™, a commercially available ECM derived from mouse cancer cell-lines that allows culturing stem cells without inducing differentiation, and many forms of polyethylene glycol- based hydrogels.

[0004] Control over the pore dimensions of hydrogels allows control over the permeability properties of the hydrogels and their adaptability to various applications. For example, when hydrogels are used to encapsulate cells for transplantation purposes, the porosity is an important parameter that has a direct role in the diffusion of molecules through the hydrogels and in the protection of encapsulated cells against the immune system. Other factors that are important in designing hydrogels for implantation are how they interact with tissues at their implantation site. Various aspects need to be optimized, including minimization of inflammatory responses that could lead to fibrotic overgrowth of the hydrogel, causing hypoxia of the encapsulated therapeutic cells. Another consideration involves mechanical resilience of the hydrogels, which ideally are designed to persist for extended periods of time in vivo. Accordingly, improvements in hydrogels are desired, particularly with respect to the control of their porosity and thus permeability to components of the immune system, and their ability to retain cells.SUMMARY

[0005] In one aspect, there is provided a method of printing a hydrogel patch containing live cells, the method comprising: extruding a mixture comprising alginate, a first polymer, a second polymer, and live cells through a needle or nozzle, wherein the first polymer is functionalized with a homocysteine thiolactone of formula I, wherein any three of the R2 are H, and the remaining R2 is selected from H, C1-C4 branched or linear alkyl, methoxy, ethoxy, and halogen;wherein the second polymer is functionalized with a pendant vinyl group on a second polymer; while extruding, moving the needle or the nozzle to form a precursor of the hydrogel patch; gelling the alginate, preferably by submersion of the precursor of the hydrogel in a bath containing calcium, strontium, barium, iron, or zinc ions to gel the alginate; and reacting the first polymer functionalized with the homocysteine thiolactone of formula I to open the ring and obtain a pendant thiol group, and crosslinking the pendant thiol group of the first polymer with the pendant vinyl group on the second polymer to obtain the hydrogel patch containing live cells. There is also provided a hydrogel patch obtained by the method as defined herein.

[0006] In a second aspect, there is provided a hydrogel patch comprising ionically crosslinked alginate; a polymer formed by the covalent crosslinking of by a first polymer that is functionalized with a homocysteine thiolactone of formula I, wherein any three of the R2 are H, and the remaining R2 is selected from H, C1-C4 branched or linear alkyl, methoxy, ethoxy, and halogenand a second polymer that is functionalized with a pendant vinyl group on a second polymer; and live cells encapsulated in the alginate and the polymer of the hydrogel patch.

[0007] In at least some embodiments of the first or second aspect, the mixture is extruded in layers, preferably wherein each layer has the same or substantially the same size and shape. In at least some embodiments, each layer comprises a substantially planar array of intersecting “strings” defining apertures therebetween having a surface area of 100,000 to 500,000 pm2, wherein the hydrogel precursor comprises at least two layers.

[0008] In at least some embodiments of the first or second aspect, the first polymer and / or the second polymer have a backbone selected from poly(methyl vinyl ether-alt-maleic anhydride) (PMM), homopolymer of methacrylic anhydride, homopolymer of acrylic acid, homopolymer of methacrylic acid, or copolymers of acrylic acid and methacrylic acid, or copolymers of maleic anhydride with vinyl ethers selected from methyl vinyl ether, ethyl vinyl ether, propyl vinyl ether, butyl vinyl ether, hydroxy ethyl vinyl ether, and methoxy ethyl vinyl ether, copolymers of N- vinylpyrrolidone with maleic or itaconic or citraconic anhydrides.

[0009] In at least some embodiments of the first or second aspect, the first polymer and / or the second polymer has a backbone that comprises monomeric units selected from 2-aminoethyl acrylate, 2-aminoethyl methacrylate, 2-aminoethyl acrylamide, 2-aminoethyl methacrylamide, 3- aminopropyl acrylate, 3-aminopropyl methacrylate, 4-aminobutyl acrylate, 4-aminobutyl methacrylate, 4-aminobutyl acrylamide, 4-aminobutyl methacrylamide, acrylic acid (AA), acrylamide, methacrylic acid (MAA), 2-hydroxypropylacrylamide (HPA), 2- hydroxypropylmethacrylamide (HPMA), 2-hydroxyethylacrylate (HEA), 2- hydroxyethylmethacrylate (HEMA), 2-(methacryloyloxy)ethyl 2-(trimethylammonio)ethyl phosphate (MPC), carboxy betaine methacrylate (CBM), carboxybetaine methacrylamide, N,N- dimethylaminoethylacrylate (DMAEA), N,N-dimethylaminoethyl methacrylate, 3-N,N-dimethylaminopropyl acrylamide, 3-N,N-dimethylaminopropyl methacrylamide, 3-N,N- dimethyliaminopropyl acrylate, 3-N,N-diaminopropyl methacrylate, 4-N,N-dimethylaminobutyl acrylamide, 4-N,N-dimethylaminobutyl methacrylamide, 1 ,3-bis(dimethylamino)propyl acrylate, 1 ,3-bis(dimethylamino)propyl methacrylate, 2-((2-(dimethylamino)ethyl)(methyl)amino)ethyl methacrylate, 2-((2-(dimethylamino)ethyl)(methyl)amino)ethyl acrylate, sodium acrylate, sodium methacrylate, sodium ethacrylate, hydroxyethyl acrylate, hydroxyethyl methacrylate, [2- (acryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, 3-((3- methacrylamidopropyl)dimethylammonio)propane-1 -sulfonate, 2-(acryloyloxy)ethyl phosphate, 2-(methacryloyloxy)ethyl phosphate, (2-(methacryloyloxy)ethyl)phosphonate, (2- (acryloyloxy)ethyl)phosphonate, (2-acrylamidoethyl)phosphonate, (2- methacrylamidoethyl)phosphonate, 2-methacryloyloxyethyl phosphorylcholine, N- (carboxymethyl)-2-(acryloyloxy)-N,N-dimethylethan-1-aminium, N-(carboxymethyl)-2-(methacryloyloxy)-N,N-dimethylethan-1-aminium, N-(carboxymethyl)-3-methacrylamido-N,N- dimethylpropan-1-aminium, N-(carboxymethyl)-3-acrylamido-N,N-dimethylpropan-1-aminium, 2- (dimethylamino)ethyl acrylate, 2-(dimethylamino)ethyl methacrylate, / V-[3-(Dimethylamino)propyl] acrylamide, / V-[3-(dimethylamino)propyl] methacrylamide, / V-(3-Aminopropyl)acrylamide hydrochloride, / V-(3-aminopropyl)methacrylamide hydrochloride, vinyl sulfonic acid, vinyl phosphonic acid, 3-sulfopropyl methacrylate potassium salt, 2-(methacryloyloxy)ethanesulfonic acid, and combinations thereof.

[0010] In at least some embodiments of the first aspect, the reacting step involves or is preceded by a hydrolysis or an aminolysis.

[0011] In at least some embodiments of the first aspect, the needle or nozzle has a diameter of from 200 to 1 ,200 pm.

[0012] In at least some embodiments of the first aspect or second aspect, the mixture is an aqueous mixture comprising from 0.5 to 2 wt. % alginate, from 0.25 to 1 wt. % of the first polymer and from 0.25 to 1 wt. % of the second polymer.

[0013] In at least some embodiments of the first or second aspect, the alginate (e.g. in the mixture) is provided at a weight ratio of alginate to a total of the first and the second polymer being of 1 :to 1.1 , to 1.1 to 1.

[0014] In at least some embodiments of the first or second aspect, the first polymer and the second polymer are provided in a weight ratio of 1 : 1 .1 to 1.1 : 1 .

[0015] In at least some embodiments of the first aspect, the bath comprises up to 100 mM of a total of the calcium, strontium, barium, iron, or zinc ions.

[0016] In at least some embodiments of the first or second aspect, the pendant vinyl group is a pendant vinylsulfone, and / or all of the R2 is H.

[0017] In at least some embodiments of the first or second aspect, the method further comprises coating the hydrogel with a third polymer such as poly[APM-co-DMAEA-co-SBMA] (PADS) or the hydrogel is coated with the third polymer such as the PADS.

[0018] In at least some embodiments of the first or second aspect, the live cells include cell aggregates, preferably pancreatic islets.

[0019] There is also provided a hydrogel patch as defined herein or as obtained by the method as defined herein, and incorporating a number of islet equivalents (I EQ) ranging from 1 ,000 to 100,000. In at least some embodiments, the hydrogel patch has a surface area of its layers being of from 1 cm2to 100 cm2.

[0020] There is provided the use of the hydrogel patch as defined herein or as obtained from the method described herein, for promoting the production of insulin in an individual in need thereof.

[0021] There is provided the use of the hydrogel patch as defined herein or as obtained by the method described herein, for treating diabetes.

[0022] Still, there is provided a method of treating diabetes comprising transplanting in a subject in need thereof the hydrogel patch as defined in herein or as obtained by the method described herein. In at least some embodiments, the method further comprises explanting the hydrogel patch at least a week after transplanting in the subject. In at least some embodiments, the live cells remain alive and are beta cells that produce insulin and still produce insulin when explanted. In at least some embodiments, the hydrogel patch is implanted in the peritoneal cavity.

[0023] Many further features and combinations thereof concerning the present improvements will appear to those skilled in the art following a reading of the instant disclosure.DESCRIPTION OF THE DRAWINGS

[0024] FIG. 1 is a1H nuclear magnetic resonance (NMR) spectrum of polymethyl methacrylate homocysteine thiolactone (PMM-HTL) in D2O.

[0025] FIG. 2 is a1H NMR spectrum of poly[APM-co-DMAEA-co-SBMA] in D2O.

[0026] FIG. 3 is a schematic showing the formation of crosslinked capsules from PMM-HTL and PMM-cysteamine vinyl sulfone (PMM-CVS).

[0027] FIG. 4 is a schematic showing illustrations of hydrogel capsules, strings, or patch filaments with thiolactone groups used in three example configurations. Homocysteine thiolactone groups, depicted as pentagons, can be incorporated within the hydrogel (configuration 1), onto the hydrogel surface as part of a coating (configuration 2), and both within and on the hydrogel (configuration 3).

[0028] FIG. 5A is a microscopy image showing hydrogel capsules (about 1 mm diameter) formed with 2 wt.% total PMM-CVS / PMM-HTL (1 : 1 wt. ratio) with 1 % alginate, coated with 0.3 wt.% PADS (formulation labeled sample “A”).

[0029] FIG. 5B is a microscopy image showing human donor islets encapsulated in the hydrogel capsules of Fig. 5A.

[0030] FIG. 6A is a confocal microscopy image of “A” hydrogel capsules showing the fluorescein isothiocyanate (FITS) channel for PMM-CVS-FITC.

[0031] FIG. 6B is a confocal microscopy image of “A” hydrogel capsules showing the tetramethylrhodamine (TRITC) channel for PADS.

[0032] FIG. 6C is a confocal microscopy image combining the FITC and TRITC channels of Figs. 6A and 6B.

[0033] FIG. 7A is a brightfield microscopy image of “A” as formed.

[0034] FIG. 7B is a brightfield microscopy image of “A” after treatment with sodium citrate.

[0035] FIG. 7C is a brightfield microscopy image of “A” after treatment with sodium citrate and then sodium hydroxide.

[0036] FIG. 8 is a microscopy image showing hydrogel capsules formed with 4 wt.% total PMM-CVS / PMM-HTL (1 :1 wt ratio) with 1 % alginate, coated with 1 wt.% PADS (formulation labeled sample “B”).

[0037] Fig. 9 is a microscopy image showing islets encapsulated in “B” of Fig. 8.

[0038] FIG. 10 is a microscopy image showing hydrogel capsules formed with 2 wt.% totalPMM-CVS / PMM-HTL (1 :1 wt ratio) with 1 % alginate, coated with 1 wt.% PADS with 25 kDa instead of 15 kDa (formulation labeled sample “C”).

[0039] FIG. 11A is a confocal cross-sectional image of C capsules during lgG-alexafluor488 in-diffusion study after about 24 h incubation with the TRITC-channel showing PADS coating forming the permeability-reducing membrane in the outer capsule shell.

[0040] FIG. 11 B is a confocal cross-sectional image of C capsules during lgG-alexafluor488 in-diffusion study after about 24 h incubation which combines Fig. 11 A with FITC-channel showing lgG-alexafluor488 largely excluded from capsule.

[0041] FIG. 12 is a graph showing the in-diffusion of IgG across hydrogel capsules after 24 hours (control (Ctrl) is an uncoated hydrogel control capsule).

[0042] FIG. 13 is a confocal cross-sectional image of capsules (formulation “D”) taken in the TRITC channel showing thiolactone containing PHMS polymer as the final coating.

[0043] FIG. 14 is a graph showing the diffusion of IgG antibodies across capsules (blank or containing human donor islets (formulation “E”)).

[0044] FIG. 15 is a microscopy image showing “D” capsules containing rat islets that were retrieved after implantation in diabetic rats.

[0045] FIG. 16A is a brightfield image of a coated hydrogel patch formulation.

[0046] FIG. 16B is a stitched fluorescence image of the coated hydrogel patch formulation, showing fluorescence of the PADS coating (TRITC channel).

[0047] FIG. 17A is a confocal cross-sectional image of the coated hydrogel patch, showing the PADS polymer coating in the TRITC channel.

[0048] FIG. 17B is a brightfield cross-sectional image of the coated hydrogel patch.

[0049] FIG. 18A is a brightfield cross-sectional image of the coated hydrogel patch after treatment with sodium citrate followed by sodium hydroxide.

[0050] FIG. 18B is a confocal microscopy image of the coated hydrogel patch of Fig. 18A.

[0051] FIG. 19A is a fluorescence microscopy image showing the live staining results of islets in a patch sized for rat implants(1.4 cm x 1.2 cm) one day after encapsulation.

[0052] FIG. 19B is a fluorescence microscopy image showing the dead staining results of islets in a patch sized for rat implants(1.4 cm x 1.2 cm) one day after encapsulation.

[0053] FIG. 19C is a composite image of Fig. 19A and Fig. 19B.

[0054] FIG. 20A is a graph showing the Glucose Stimulated Insulin Secretion (GSIS) results of islet-laden patches on day 3 after implantation in rats.

[0055] FIG. 20B is a graph showing the GSIS results of islet-laden patches on day 5 after implantation in rats.

[0056] FIG. 21 is a graph showing the results of dextran-fluorescein (70K) diffusion across the double coating and into the alginate / PMM-thiol gel core after 24 hours.

[0057] FIG. 22A is a fluorescence microscopy image showing Dextran-fluorescein (70K) diffusion where one of the coating polymers is rhodamine-labeled and the coating was applied at a 25 mM CaCh concentration after 24 hours (here showing Dextran channel).

[0058] FIG. 22B is a fluorescence microscopy image showing Dextran-fluorescein (70K) diffusion where one of the coating polymers is rhodamine-labeled and the coating was applied at a 25 mM CaCh concentration after 24 hours (here showing rhodamine channel).

[0059] FIG. 22C is a brightfield microscopy image of Figs 22A-22B.

[0060] FIG. 23A is a fluorescence microscopy image showing Dextran-fluorescein (70K) diffusion where one of the coating polymers is rhodamine-labeled and the coating was applied at a 50 mM CaCh concentration after 24 hours (here showing Dextran channel).

[0061] FIG. 23B is a fluorescence microscopy image showing Dextran-fluorescein (70K) diffusion where one of the coating polymers is rhodamine-labeled and the coating was applied at a 50 mM CaCh concentration after 24 hours (here showing rhodamine channel).

[0062] FIG. 23C is a brightfield microscopy image of Figs 22A-22B.

[0063] FIG. 24 is a photograph of a patch of 4.5 x 4.0 cm 3-layer print design that had a triangular infill pattern with a 34% infill density and an overall volume close to 1 mL.

[0064] FIG. 25A is a brightfield microscopy image of an oval patch in the gelling bath on the day it was printed.

[0065] FIG. 25B is a photograph of the oval patch of Fig. 25A implanted in a rat model (after 4 weeks).

[0066] FIG. 26A is a combined brightfield microscopy image and fluorescence microscopy image showing a conformally coated islet.

[0067] FIG. 26B is the fluorescence microscopy image only of Fig. 26A.DETAILED DESCRIPTION

[0068] There is provided a hydrogel for producing capsules, strings, patches and the like but most preferably patches. In this context, a patch refers to 3-dimensional array of connected hydrogel strings. The hydrogel is crosslinked with thiol-ene chemistry between two polymers, one functionalized with a thiol group and one functionalized with a vinyl group. The polymer functionalized with a thiol group has a pendant thiol group, such as a homocysteine as shown below where X can be a primary or secondary amine, a hydroxyalkyl, a hydroxyl or -O'. X can be linked to a polymer backbone.polymer backbone

[0069] Although the present disclosure provides examples with the backbone polymers being poly(methyl vinyl ether-alt-maleic anhydride) (PMM), other alternatives are possible. Indeed, alternatives to PMM include but are not limited to other copolymers of maleic anhydride with other vinyl ethers (e.g. ethyl vinyl ether, propyl vinyl ether, butyl vinyl ether, hydroxyethyl vinyl ether, and methoxy ethyl vinyl ether). Other alternatives include itaconic anhydride or citraconic anhydride with vinyl ether comonomers such as methyl vinyl ether, ethyl vinyl ether, propyl vinyl ether, butyl vinyl ether, hydroxyethyl vinyl ether, and methoxy ethyl vinyl ether, and copolymers of N-vinylpyrrolidone with maleic or itaconic or citraconic anhydrides. In some embodiments, the backbone polymer is a homopolymer of methacrylic anhydride, acrylic acid, methacrylic acid, or a copolymer of acrylic acid and methacrylic acid.

[0070] To obtain the pendant thiol group, a cysteamine thiolactone group is first functionalized onto the polymer and then the ring is opened via hydrolysis or aminolysis. In one example, a thiolactone group was functionalized onto PMM by reaction of the amino group of homocysteine thiolactone (HTL) with the anhydride groups of PMM. The homocysteine thiolactone functionalized PMM polymers (PMM-HTL) can then undergo a ring opening reaction to obtain PMM-homocysteine having reactive free thiol groups. One example of a scheme to functionalize PMM with homocysteine thiolactone is presented below.Scheme 1.

[0071] The term “homocysteine thiolactone” or “HTL” as contemplated by the present disclosure, is of formula I where three of the R2 are H, and the remaining R2 is selected from H, C1-C4 branched or linear alkyl, methoxy, ethoxy, and halogen.

[0072] Accordingly, in some embodiments, PMM-homocysteine as per formula Ila or lib below is used in the formation of hydrogels. X is as defined above, or preferably X is O' or NHR where R is a C1-C4 alkyl. It should be noted that R can be a linker or directly linked to a backbonepolymer which could be PMM or a polyacrylate or polymethacrylate polymer bearing primary amine groups.

[0073] When the ring opening is performed by aminolysis, X is NHR:

[0074] Aminolysis can involve small amines, but preferably large molecules and most preferably polymers containing multiple amine groups. These polyamines are electrostatically attracted to the anionic HTL polymer, and by virtue of their relatively high amine density are likely to contain a mixture of protonated and non-protonated primary amines, even at physiological pH, due to the well known polyelectrolyte effect. These two effects: electrostatic attraction and presence of non-protonated primary amines, can speed aminolysis and hence the overall crosslinking reaction.

[0075] The ring opening by aminolysis is performed based on the following scheme:Scheme 2

[0076] On the other hand, when the ring opening is performed by hydrolysis, X in formula lib is O' or O-H (as shown in the structure below). Hydrolysis and subsequent crosslinking of the generated thiol can be performed in vitro in presence of 10% serum, and therefore can also proceed in vivo under similar conditions. In one example, the capsules can crosslink in vitro over a span of two hours or longer while at pH 7.4-7.6 in buffered solutions. This time may be shortened by carrying out this reaction at slightly elevated temperatures such as 37 °C. These time spans work well with typical time periods between encapsulation and implantation. As usual, care must be taken to ensure proper oxygenation of encapsulated cells during this period between encapsulation and implantation, through means known to people skilled in the art. Hydrolysis and / or aminolysis, together with subsequent crosslinking, may continue even after implantation in vivo.

[0077] The pendant thiol group can react with a vinyl group to form a crosslinked hydrogel. Preferred vinyl groups are vinylsulfones. This is because vinylsulfones are strongly activated electrophiles, able to react with accessible thiol-functional and amine-functional groups on, e.g polymers such as PMM-homocysteine. While pendant vinylsulfone groups on polymer backbones deactivate by spontaneous hydration to form 2-hydroxyethylsulfone groups, a hydrophilic and largely benign group, some of the residual vinylsulfone groups can also be used to bind other thiol- and amino- functional groups, such as cysteine, mercaptoethanol, and cysteamine to modify hydrophilicity and charge density of the hydrogel for example.

[0078] Accordingly, although the present disclosure provides PMM-cysteamine vinyl sulfone (PMM-CVS) as an example of vinyl group, this is not a limitative example as not only polymers other than PMM can be used, but also other pendant activated vinyl or preferably vinylsulfone groups can be used. A preferred embodiment, which is exemplified in the Example section below, is the formation of a hydrogel between PMM-cysteamine vinyl sulfone (PMM-CVS) and PMM- homocysteine thiolactone (PMM-HTL) together with a separate initial gel former. The initial gel former is for example alginate, hyaluronic acid, gelatin, hydroxypropylcellulose, carboxymethylcellulose, methylcellulose, pluronic, or elastin. The initial gel former can be provided in a weight percentage of from 0.5 to 10 wt. %, and preferably 1 to 4 wt. %, and most preferably 1 .5 to 3 wt. %, depending on the viscosity and the type of initial gel former. If the initial gel former is alginate, it is mixed with the synthetic gel formers, and the overall gel is formed by a one-pot two-stage process where the mixture (optionally containing cells) is extruded into a bath containing calcium, strontium, barium, iron, or zinc ions that rapidly gel the alginate to providestructural integrity with ionic crosslinking while the thiolactone covalent crosslinking takes place over the subsequent time of handling, washing steps until implantation, on the order of typically one to three hours.

[0079] The chemical formula of PMM-CVS is presented below:

[0080] As explained above, it should be understood that other vinylsulfone groups and other polymer backbones are suitable. For example, the vinyl functional polymer is more generally a cysteamine vinylsulfone modified polymer as shown below:Polymer backbone

[0081] PMM-homocysteine generated from PMM-HTL as shown in Scheme 2 reacts with PMM-CVS, a thiol-ene reaction between the pendant thiol of PMM-homocysteine and the pendant vinyl group of PMM-CVS to form the structure shown below (X is as defined as a primary, secondary amine, a hydroxyalkyl, a hydroxyl or -O'):

[0082] The aminolysis of thiolactone groups has also been used here to covalently crosslink polycations while coating them onto hydrogel capsule cores (gels). In the context of the present disclosure the term “coat” or “coating” refers to the coating of a layer or the grafting of chemical species or functional groups on a surface, for example the shell of a particle. This process involves reaction of primary or secondary amines on these polycations, with residual homocysteine thiolactone groups on the core-hydrogel, and thus allows to post-modify capsules formed from PMM-CVS and PMM-HTL for example, with primary-amine functional copolymers. In addition to polycations, this process may also be carried out with polyampholytes comprising primary or secondary amines. The polyampholytes can be stoichiometric (50-50 anionic and cationic), close to stoichiometric (45-55 or 55-45 anionic to cationic respectively) or non-stoichiometric (70% cationic and 30 % anionic). This approach allows improved control over final pore-size and hydrogel exclusion properties. It also optionally enables introducing immune-modulating functions into the shell of the hydrogel. Finally, it would provide another route to reinforce the outer shell, to enhance mechanical resilience and cell retention. These benefits would similarly apply to other hydrogel geometries such as strings and patches comprising this hydrogel chemistry.

[0083] One of the polycations that was developed for coating hydrogels is a poly[APM-co- DMAEA-co-SBMA] terpolymer (PADS). APM is 3-aminopropylmethacrylamide which contains a primary amine to react with a thiolactone. DMAEA is N,N-dimethylaminoethylacrylate and SBMA is [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide. A key formulation is the PADS112 series of ternary copolymer, where 112 or 1 :1 :2 means ternary copolymer, where 1 :1 :2 represents initial molar feed ratios of 1 :1 :2 of APM, DMAEA, and SBMA. The molar ratios can vary by ±50%, ±20%, ±10%, ±5% or ±3% each, and additional comonomers can be contemplated. In one example, to obtain the 1 : 1 :2 ratio a set of copolymers with molecular weights (Mn) of 15,000, 25,000 and 40,000 g / mol, respectively APM, DMAEA and SBMA were prepared using reversible addition-fragmentation chain-transfer (RAFT) polymerization. The three comonomers have significantly different reactivity ratios, which introduces drift during copolymerization - however, due to the controlled (‘living’) nature of this polymerization, this is translated into gradient copolymer chains where each chain has more SBMA at the initiator end, and more cationic comonomers at the other end. An advantage of the gradient distributions is that it facilitates electrostatic binding and covalent reaction of the cation-rich end of the polymer to the anionic hydrogel, while leaving the SBMA-rich end to be tissue-facing. The amine of APM contributes reactivity as well as cationic density for coating onto anionic PMM-CVS / PMM-HTL core hydrogels. DMAEA (N,N-dimethylaminoethyl acrylate) has a tertiary amine in the pendantgroup which also contributes cationic charge density to assist with coating onto capsules. DMAEA has the additional benefit that it undergoes charge-shifting by hydrolysis with a half-life measured in hours to days. As a result, its initial cationic charge density facilitates binding to capsule and the anionic, HTL-functional polymer network, but after covalent crosslinking and implantation, its charge density decreases by hydrolysis of the DMAEA group and loss of dimethylaminoethanol. This process reduces the overcall cationic charge density of the shell of the capsule and improves anti-fouling properties. Finally, SBMA is [2-(methacryloyloxy)ethyl]dimethyl-(3- sulfopropyl)ammonium hydroxide, a zwitterionic monomer that further improves the antifouling properties of the coating by increasing the hydrophilicity of the materials. The structure of PADS is shown below where n, m, and x represent repeating subunits (that can be a statistical distribution and not necessarily in the order shown in the structure below).

[0084] With PADS which has a primary amine to react as the pendant amine (NHa+) for the aminolysis, the structure of PMM-HTL - PMM-CVS - PADS is as follows:

[0085] A polymer derived of PADS was also developed by excluding SBMA, namely poly[3- aminopropylmethacrylamide-co-2-(dimethylamino)ethyl acrylate] (PAD). PAD retains the advantage of APM and DMEAE as explained above.(that can be a statistical distribution and not necessarily in the order shown in the structure below).

[0086] A PMM-HTL - PMM-CVS - PAD can also be produced as explained above for PADS and would have the following structure

[0087] Polycations other than PADS and PAD are also contemplated herein for the coating of the hydrogels, for example other copolymers with primary amino containing comonomers like APM, such as 2-aminoethyl acrylate, 2-aminoethyl methacrylate, 2-aminoethyl acrylamide, 2- aminoethyl methacrylamide, 3-aminopropyl acrylate, 3-aminopropyl methacrylate, 4-aminobutyl acrylate, 4-aminobutyl methacrylate, 4-aminobutyl acrylamide, 4-aminobutyl methacrylamide, acrylic acid (AA), acrylamide, methacrylic acid (MAA), 2-hydroxypropylacrylamide (HPA), 2- hydroxypropylmethacrylamide (HPMA), 2-hydroxyethylacrylate (HEA), 2- hydroxyethylmethacrylate (HEMA), 2-(methacryloyloxy)ethyl 2-(trimethylammonio)ethyl phosphate (MPC), carboxy betaine methacrylate (CBM), carboxybetaine methacrylamide, N,N- dimethylaminoethylacrylate (DMAEA), N,N-dimethylaminoethyl methacrylate, 3-N,N- dimethylaminopropyl acrylamide, 3-N,N-dimethylaminopropyl methacrylamide, 3-N,N- dimethyliaminopropyl acrylate, 3-N,N-diaminopropyl methacrylate, 4-N,N-dimethylaminobutyl acrylamide, 4-N,N-dimethylaminobutyl methacrylamide, 1,3-bis(dimethylamino)propyl acrylate, 1 ,3-bis(dimethylamino)propyl methacrylate, 2-((2-(dimethylamino)ethyl)(methyl)amino)ethyl methacrylate, 2-((2-(dimethylamino)ethyl)(methyl)amino)ethyl acrylate, sodium acrylate, sodiummethacrylate, sodium ethacrylate, hydroxyethyl acrylate, hydroxyethyl methacrylate, [2- (acryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, 3-((3- methacrylamidopropyl)dimethylammonio)propane-1 -sulfonate, 2-(acryloyloxy)ethyl phosphate, 2-(methacryloyloxy)ethyl phosphate, (2-(methacryloyloxy)ethyl)phosphonate, (2- (acryloyloxy)ethyl)phosphonate, (2-acrylamidoethyl)phosphonate, (2- methacrylamidoethyl)phosphonate, 2-methacryloyloxyethyl phosphorylcholine, N- (carboxymethyl)-2-(acryloyloxy)-N,N-dimethylethan-1-aminium, N-(carboxymethyl)-2-(methacryloyloxy)-N,N-dimethylethan-1-aminium, N-(carboxymethyl)-3-methacrylamido-N,N- dimethylpropan-1-aminium, N-(carboxymethyl)-3-acrylamido-N,N-dimethylpropan-1-aminium, 2- (dimethylamino)ethyl acrylate, 2-(dimethylamino)ethyl methacrylate, / V-[3-(Dimethylamino)propyl] acrylamide, / V-[3-(dimethylamino)propyl] methacrylamide, / V-(3-Aminopropyl)acrylamide hydrochloride, / V-(3-aminopropyl)methacrylamide hydrochloride, vinyl sulfonic acid, vinyl phosphonic acid, 3-sulfopropyl methacrylate potassium salt, and 2- (methacryloyloxy)ethanesulfonic acid and combinations thereof.

[0088] The overall composition, net cationic charge, and molecular weight of the polycation used are key parameters for hydrogel coatings. Where the hydrogel is coated with a single coating polymer, the composition of the polycation is preferably 30-80 mol% net cationic in charge, with the remaining 70-20 mol% of the coating polymer containing hydrophilic, charge-neutral comonomer. Of the 30-80 mol% cationic charge, preferably at least 20 mol% is primary amine containing, with the remaining cationic charge from comonomers containing tertiary or quaternary and optionally charge-shifting ammonium cations. Molecular weight of the polycation is also key to coating the hydrogels and forming permeability-controlling membranes, and can be 5,000- 50,000 g / mol, but more preferably 15,000-40,000 g / mol.

[0089] The use of small molecule amines such as lysine, glycinamide, and the like as reagents to ring open thiolactone groups was tested. However, it was found that the rate of aminolysis using small molecule amines at physiologically reasonable concentrations was relatively slow, and became a limiting factor for feasibility of the crosslinking timeline, given encapsulation of cells. Accordingly, although using small amines is feasible, it is preferable, especially for large scale processes, to use larger amines such as polymers containing multiple amine groups. Without wishing to be bound by theory, it is hypothesized that the pKa of small molecules amines (pKa ~10) are a limiting factor for aminolysis, especially when the pH for hydrogels comprising cells is typically buffered at pH 7.4.

[0090] One advantage of utilizing polycation coatings is that the polycations are held in place electrostatically which facilitates reaction with thiolactone groups bound on polyanionic polymers including but not limited to PMM. Within a polycation, ammonium groups may have a range of pKa’s (pKa 7-10), significantly lower than the pKa of their small molecule analogues, which also may facilitate the aminolysis reaction with the thiolactones.

[0091] Following the initial coating of the core hydrogel with primary or secondary amine containing polycations, the surface of the hydrogels have a net cationic charge and can thus be coated sequentially with a polymer with a net anionic charge (polyanion). This layer-by-layer coating of the hydrogels with (1) net polycations followed by (2) polyanions can be repeated to form hydrogel membranes with increasing polymer density upon each added layer. It is important to recognize that, while these processes are called coating processes in the art, the polymers used in these coatings do not actually add to the surface of these very permeable hydrogel, but are more typically absorbed into the outer layers of the hydrogel. As a result, these coating processes do not tend to increase the diameter of the hydrogel, but rather increase density of the outer layers. Further, both initial hydrogel components and coating polymers coexist in these outer layers, and, given correct chemical compositions described herein, may react with each other both ionically and covalently. This concept is key to using reactive polycation / polyanion pairs to coat, modify permeability to effect tighter molecular weight exclusion, and otherwise reinforce the outer layers of these hydrogels. The number of layers, in addition to the concentration of the polymer in each layer, can therefore be used as another method to control the overall permeability of the hydrogel coating. In one embodiment, the thiolactone functional polymers can be utilized to layer-by-layer coat cells and cell clusters directly to form covalently crosslinked conformal coatings. Cells can be coated with the polycation to electrostatically bind to the net anionic surface of the cells. These polycation coated cells can then be rinsed to remove excess unbound polycations, followed by exposure to thiolactone functional net polyanion, such as PHMS-60-25k. The resulting initial electrostatic complex between polycation and polyanion will spontaneously form covalent crosslinks to each other, to form a permanently crosslinked membrane on the surface of the cells. This layer-by-layer approach can be repeated to increase the thickness and density of the conformal coating.

[0092] A preferred polyanion for use as second coating during layer-by-layer coating of the hydrogels was similarly designed to contain thiolactone groups. This allows for covalent crosslinking by reaction with the primary amine of the previous, polycation, layer. This ringopening of thiolactone generates free thiol groups, which can further react with residualvinylsulfone present as part of the core hydrogel. Similarly, some of the primary amine groups from the polycation coating may react with residual HTL-functional groups from the core hydrogel to form crosslinks as well as further thiol groups. These mechanisms hence can form crosslinks between the two coating materials (polycation and polyanion), but also between the polycation coating with core-HTL-polymer. Finally, any polymer-bound thiol formed in core or shell can react with vinylsulfone groups present in the outer layers of the core hydrogel. The homocysteine thiolactone (HTL)-containing polyanions are prepared as described as follows. This multiplicity of simultaneous crosslinking is illustrated in Fig. 4.

[0093] Homocysteine thiolactone may be formed into a monomer via reaction with (meth)acryloyl chloride or (meth)acrylic anhydride to form homocysteine thiolactone (meth)acrylamide (HTL(M)A). Homocysteine thiolactone monomers can be copolymerized to generate homocysteine thiolactone based polymers that can be used both as gel formers, similar to PMM-HTL, in addition to polymers for coatings. HTL(M)A can be homo- and copolymerized with anionic comonomers such as (meth)acrylic acid ((M)AA), 2-acrylamido-2-methyl-1 -propane sulfonic acid, 3-sulfopropyl methacrylate, and zwittionic comnomers such as SBMA, MPC, and carboxybetaine methacrylates (CBMA) as well as other betaines. Homocysteine thiolactone based polymers with a net anionic charge can be used as a coating polymer to coat hydrogels with a net cationic surface charge, such as polycation coated hydrogels. Thus, these HTL functional polymers can also be covalently crosslinked to surfaces and coatings that have primary amino groups, in addition to forming core hydrogel constructs.

[0094] As HTL functional polyanions can be coated as the final layer on hydrogels, these HTL(M)A copolymers can be designed to incorporate functional groups, biomolecules and factors to modify the surface chemistry of the coated hydrogel. HTL(M)A can be copolymerized with monomers such as neutral hydrophilic monomers including SBMA, MPC, carboxybetaine methacrylates (CBMA), and hydroxyethyl(meth)acrylate (HEMA) to create an anti-fouling surface when coated onto the hydrogel by creating a hydration shell, in addition to anionic comonomers to allow electrostatic interactions with polycations containing primary amino groups and to allow covalent crosslinking with the homocysteine thiolactone groups.

[0095] The molecular weight (MW) of these HTL functional coating polymers can be varied to control the penetration depth and therefore thickness of the coating. The lower MW HTL copolymers are able to diffuse more into the hydrogel and electrostatically and covalently bind to a larger volume of polycation coated hydrogel, thus creating thicker crosslinked coatings. HigherMW HTL copolymers penetrate less into the hydrogel, and thus create thinner crosslinked coatings.

[0096] Homocysteine thiolactone may also be formed into a monomer via reaction with (meth)acryloyl chloride or (meth)acrylic anhydride to form homocysteine thiolactone (meth)acrylamide (HTL(M)A). The reaction scheme to form the homocysteine thiolactone methacrylamide monomer is as follows:

[0097] The reaction scheme to form homocysteine thiolactone acrylamide is as follows:

[0098] The monomers homocysteine thiolactone methacrylamide and homocysteine thiolactone acrylamide can be used to produce a homocysteine thiolactone polymer as an alternative route to functionalization of polymer backbones to include the homocysteine thiolactone groups. The two monomers can be employed as sole monomers (homopolymerization) or with each other (copolymerization). The homocysteine thiolactone polymer is obtained by radical-mediated polymerization aided by a radical initiator, optionally under heating. One example of a radical initiator is azobisisobutyronitrile (abbreviated AIBN) which can be operated in organic solvents (e.g. tetrahydrofuran, toluene, dioxane, ethylacetate, chloroform) at 60-80 °C. Vazo™ initiators such as Vazo-56TMcan be used in aqueous solvents for a controlled radical polymerization. In another example, V501™ radical initiator can be used.

[0099] Polymerization of homocysteine thiolactone monomers under aqueous conditions and with amine-containing components is compatible at low pH, preferably in the range of pH 3-4, where hydroxide concentration is low and amine functional groups are in the cationic ammonium form where they are non-nucleophilic.

[0100] It is important to note that a wide variety of polymer compositions can be prepared by using various comonomers, neutral, zwitterionic, anionic, and cationic comonomers. Examples of neutral comonomers include but are not limited to 2-hydroxyethyl (meth)acrylate, 2-hydroxyethyl (meth)acrylamide, 2-hydroxypropyl (meth)acrylamide, 3-hydroxypropyl (methacrylamide), polyethyleneglycol methacrylates, and methyl (meth)acrylate. Examples of zwitterionic comonomers include but are not limited to 2-methacryloyloxyethyl phosphorylcholine (MPC), [2- (methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide] (SBMA), and carboxybetaine methacrylates. Examples of cationic comonomers include but are not limited to N,N-dimethylaminoethylacrylate (DMAEA), N,N-dimethylaminoethyl methacrylate, 3-N,N- dimethylaminopropyl (meth)acrylamide, 3-N,N-dipropylaminopropyl (meth)acrylate, 4-N,N- dimethylaminopropyl (meth)acrylamide, 1 ,3-bis(dimethylamino)propyl (meth)acrylate, 2-((2- (dimethylamino)ethyl)(methyl)amino)ethyl (meth)acrylate, and combinations thereof.

[0101] In the case of copolymerization, preferred compositions include from 5 to 40 mol% of homocysteine thiolactone (meth)acrylamide. The composition of the other comonomers can be adjusted to tune anti-fibrotic properties if these HTLMA copolymers are used as antifibrotic coatings. For example, 20-70 mol% zwitterionic comonomer can be employed for preferred compositions for anti-fibrotic properties may be incorporated. In some embodiments, the copolymer also incorporates 10-40 mol% of anionic comonomer for polymers that are used to coat cationic surfaces.

[0102] Homocysteine thiolactone monomers can be copolymerized to generate homocysteine thiolactone based polymers that can be used both as gel formers, similar to PMM- HTL, in addition to polymers for coatings. HTL(M)A can be homo- and copolymerized with anionic and zwitterionic monomers such as (meth)acrylic acid, 2-acrylamido-2-methyl-1-propane sulfonic acid, 3-sulfopropyl methacrylate, -[2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide] (SBMA), methacryloyloxyethyl phosphorylcholine (MPC), and carboxybetaine methacrylates (CBM) as well as other betaines. Homocysteine thiolactone based copolymers with a net anionic charge can be used as a coating polymer to coat hydrogels with a net cationic surface charge, such as polycation-coated hydrogels. These HTL-functional polymers can also be covalently crosslinked to surfaces and coatings that have primary amino groups. A reaction scheme to form homocysteine thiolactone functional polymers through (co)polymerization is provided for poly[homocysteine thiolactone methacrylamide-co-methacrylic acid-co-[2- (methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide] (PHMS) is provided as an example as follows:

[0103] The present hydrogels are particularly suited as a means to encapsulate cells for in vivo applications by isolating the transplanted foreign cells from the immune system. Achieving immuno-isolation of encapsulated cells remains a complex task, and requires generally (i) sufficient mechanical stiffness for reliable handling during implantation, (ii) diffusion across the hydrogel with a molecular weight cut-off of around 150,000 Daltons, expressed as percent in vitro exclusion of IgG after 24hrs exposure to an external solution of IgG, (iii) the ability to physically exclude cellular and molecular immune components, and to prevent escape of the therapeutic cells, (iv) for patches, the ability to vascularize the outer and inner surfaces of the device in order to ensure both good long-term oxygen supply as well as rapid blood sugar sensing and correction. The presently described hydrogels also have the advantage of a covalently crosslinked network that provides long-term stability in vivo. The present hydrogels also have the ability to introduce additional modifications into the device surface, including but not limited to passive immune evasive groups as well as active immune modification groups. Furthermore, the present hydrogels can accommodate a high cell loading capacity coupled with high total surface area / islet equivalent to maximize metabolic exchange while maintaining on average a 100 pm stand-off distance between encapsulated cells and host tissue on average. One embodiment of encapsulated cells in the present hydrogels in the form of a printed patch is referred to as hydrogel patches. One example of hydrogel patches is described in details in the example section below.

[0104] The hydrogel patches can be printed in a variety of size and shapes, for example they can have 1 - 3 printed layers, be of oval shape, and have dimensions of 6 by 5 cm, 5 by 4 cm, or 6 by 4 cm, which is sufficient to encapsulate a therapeutic load of beta cells in one or multiple implantable hydrogel patches.

[0105] It should be noted that multiple materials can be co-printed in the hydrogel patches, including different cell types for therapeutic purposes. In the case of multiple cell types, they canbe incorporated into a single material, separated into multiple materials, or some combination thereof.

[0106] Hydrogel patches may further include materials (either cell-containing or cell-free) that confer the overall device with increased resistance to deformation, including during handling and implantation. These include inner layers or perimeters printed using a hydrogel of higher solids loading, as well as dome-shaped devices.

[0107] For enhancement of strength, stiffening additives such as cellulose nanofibers that are chemically modified so as to covalently integrate with the other gel formers are optionally added. Additionally, these additives may be functionalized with components to enhance cell viability and function or induce more rapid vascularization (i.e. RGD peptide or gastrin functionalization).

[0108] Therapeutic islet cells can be mixed with the gel formers at concentrations of 1,000 - 100,000, and preferably 10,000 to 50,000 lEQ / mL to form the hydrogel patches.

[0109] Although pancreatic islets are the main example contemplated herein, there are other stand-alone therapeutic cells that may include mesenchymal stromal cells (MSC), hepatocytes or other endocrine cells. As well, supportive cells including MSCs, endothelial cells, vascular microvessels, fibroblasts, etc., can be added to enhance the health and function of implanted islets, encourage vascular ingrowth into the implanted hydrogel patch, or both.

[0110] The overall design of the patch can have a filament size between 200 pm and 1200 pm, preferably between 300 pm - 700 pm, an infill density between 10% and 80%, preferably from 30%-70%. These parameters can be tuned to produce gaps within the structure that are roughly between 250 pm and 600 pm wide, which has been predicted to enhance natural vascularization. The infill pattern can be a rectilinear, hexagonal, or triangular grid. There may also be a perimeter on each layer made out of the same material (with the same cells) or a material to impart greater mechanical strength (with or without cells). The overall patch shape can be a rectangular prism, but, preferably, the edges are rounded. The layers themselves may also be tapered in the height of the patch as well.

[0111] Printing technologies offer multiple ways to improve the function of the printed patches. For example, multi-layer patches can be printed into shapes that facilitate rolling the patch along one axis, facilitating laparoscopic administration to the desired implantation site followed by unrolling. The hydrogel patches can be given dome-shaped structures by stepping the z-dimension upwards towards the interior of the hydrogel patch, effectively turning it into a flat dome that is much more resistant to folding deformations and other distortions. Some designs may facilitate implantation. For example, if printed with a 3,- 4- or, preferably, 6-axis printhead, the structure can have a curved, dome or bowl-like structure that may fit better into a specific intended implantation site, as well as further supporting the structure and limiting the chance of the patch deforming during handling, implantation or long-term in vivo implantations. Further, the patch can be designed and sized for implantation into specific animal models, including in a rat intraperitoneal cavity, within fascia outside of the peritoneal cavity, or subcutaneously.

[0112] It should be noted that the hydrogel patches may shrink up to approximately 25% during gelation, and the degree of shrinking needs to be taken into account within the design.

[0113] The printing can be carried out with a supporting bath system, which can be commercially supplied LifeSupport™ (Sigma Aldrich), Pluronic F127, Carbopol™, or other materials such as synthetic microgels formed by precipitation polymerization and subsequent functionalization. In particular, combining different sizes of microgels with FRESH particles can result in smoother surfaces of the printed hydrogels as well as better transparency of the printing bath, and may help recover the printed hydrogel from the printing bath.

[0114] Coating polymers promotes the exclusion of proteins that could otherwise harm transplanted cells or trigger a detrimental immune response, while still allowing sufficient diffusional transfer of oxygen, glucose, insulin, and metabolic waste products. These coating may also strengthen the mechanical characteristics of the hydrogel patches as a whole, mitigate immune recognition, and prevent cell escape.

[0115] The printed structures, along with therapeutic cells, may also include materials that could encourage vascular ingrowth. These could include:• Growth factors such as one or more of VEGF, PDGF, Ang, HGF, IGF, TNF-a, FGF-2, IL- 6, SCF, etc.,• Platelet-rich plasma components,• Immature vascular cells such as SC-derived endothelial cells, smooth muscle cells,Microvessels isolated from adipose or pancreatic tissues,• Cell spheroids consisting of, or incorporating, endothelial cells, MSCs, fibroblasts in form of cell spheroids / organoids that are less than 1mm in diameter, preferably less than 400 pm.

[0116] These entities may be included into the printed hydrogel structure, may be coated onto the structure in a wash after printing, and / or be included in an additional hydrogel structure that encases the printed material.

[0117] The printed hydrogel patch, after all post-processing steps, can be placed in a mould slightly larger than the printed structure and the encompassing hydrogel can be subsequently formed around it in the mould.

[0118] The encompassing hydrogel may optionally contain (potentially recombinant human) collagen I, collagen III, collagen IV, laminin, fibrin, hyaluronic acid, etc., and combinations thereof.

[0119] If the encompassing hydrogel is viscous enough or is shear thinning enough, the encompassing hydrogel (and any vascular components, i.e. microvessels) may be used as the support bath. This could allow for multimaterial printing with both the hydrogel precursor polymers and cells and a pro-angiogenic ink with more concentrated quantities of pro-angiogenic factors or microvessels.

[0120] This mold may contain an array of pins, to reserve space for improved post-implant penetration of vasculature into the hydrogel patch.

[0121] It is also possible to graft immuno-modulating features on the crosslinked networks. This includes methods to functionalize one of the polymer coating materials with Biotin, expose to an excess of Streptavidin, and finally exposing to another excess of biotinylated biomaterials.

[0122] Accordingly, there is provided a hydrogel patch comprising ionically crosslinked alginate; a polymer formed by the covalent crosslinking of a first polymer that is functionalized with a homocysteine thiolactone of formula I, wherein any three of the R2 are H, and the remainingR2 is selected from H, C1-C4 branched or linear alkyl, methoxy, ethoxy, and halogenand a second polymer that is functionalized with a pendant vinyl group; and live cells encapsulated in the alginate and the polymer of the hydrogel patch.

[0123] The hydrogel is preferably produced using a 3D bioprinter. For example, a mixture comprising alginate, a first polymer, a second polymer, and live cells is extruded through a needle or a nozzle of a bioprinter. The first and second polymer are as defined above. The gelling process is completed by contacting the hydrogel with calcium, strontium, barium, iron, or zinc ions to gel the alginate. This can be done by submersion in a bath containing the ions. The bath can include the ions in a concentration of up to 200 mM, such as from 1 mM to 200 mM, from 2 mM to 100 mM, or from 10 mM to 50 mM. The submersion can be performed for a duration of from 5 mins to 30 mins, from 10 mins to 20 mins or about 15 mins ±5%. Further gelation is performed by the reaction between the pendant thiol group of the first polymer and the pendant vinyl group of the second polymer to obtain the crosslinked hydrogel patch containing live cells. As previously explained the first polymer functionalized with the homocysteine thiolactone of formula I can be reacted by hydrolysis or aminolysis to open the ring and obtain the pendant thiol group. The thiolactone covalent crosslinking may take place over the time subsequent to the ionic bath, for example during subsequent handling or washing steps until implantation, in the order of typically one to three hours.

[0124] The hydrogel patch can be made of one, two, preferably three, or more layers and the layers may have the same size and shape, or they could be different. In preferred embodiments, the size and shape of the layers is the same or at least similar so as to allow stacking the layers in a structurally sound manner. In at least some embodiments, each layer comprises a substantially planar array of intersecting “strings” or “filaments” defining apertures therebetween having a surface area of 100,000 to 500,000 pm2. The apertures may be delimited by an external surface of four different strings in the same plane. The aperture may have a rectangular or squareshape. In at least some embodiments, the aperture has a surface area of 150,000 to 450,000 pm2.

[0125] When depositing and extruding the hydrogel, partial fusion occurs at the overlap points of string segments printed. These can make vertical offsets which provide mechanical strength to the overall structure. The resulting hydrogel patches can be printed in a variety of size and shapes, for example they can have 2 - 10 and preferably 3-5 printed layers, be of oval shape (external perimeter), and have dimensions of, e.g., 6 by 5 cm, 6 by 4 cm, or 4 by 4 cm, which is sufficient to encapsulate a therapeutic load of beta cells in one or multiple implantable devices. Accordingly, in some embodiments, the patches can have a surface area of its layers being of from 1 cm2to 100 cm2.

[0126] For enhancement of strength, stiffening additives such as cellulose nanofibers that are chemically modified so as to covalently integrate with the other gel formers are optionally added. Additionally, these additives may be functionalized with components to enhance cell viability and function, or participate in covalent crosslinking with linear polymers or microgels, or induce more rapid vascularization (i.e. RGD peptide or gastrin functionalization).

[0127] Therapeutic islet cell clusters can be provided at concentrations of 1 ,000 - 100,000, and preferably 10,000 to 50,000 lEQ / mL to form the hydrogel patches.

[0128] The overall design of the patch can have a string diameter between 200 pm and 1200 pm, between 200 pm and 800 pm, or between 300 pm and 700 pm, an infill density between 10% and 80%, preferably from 30%-70%. These parameters can be tuned to produce the apertures within the structure. The aperture may be roughly between 250 pm and 600 pm wide. The apertures have been found to successfully receive vascularization and therefore enhance the diffusion in and out of the bloodstream of the live cells encapsulated in the hydrogel patch.

[0129] The nozzle or needle diameter and the movements of the nozzle or needle are therefore adapted to fulfil the aforementioned size parameters. For example, the nozzle or needle may have a diameter of between 200 pm and 1200 pm, between 200 pm and 800 pm, or between 300 pm and 700 pm. The nozzle generally operates at a speed of from 5 to 15 mm / s and a flow rate of 5 to 10 pL / s.

[0130] The density and porosity of the strings making up the hydrogel patch can be modified by modifying the concentration of alginate and the first and second polymer. In someembodiments, the mixture comprises alginate in a concentration of from 0.5 to 5 wt. %, from 0.5 to 2 wt. %, from 0.75 to 1.5 wt. % or 0.8 to 1.2 wt. %. In some embodiments, the mixture comprises a total of the first and second polymer alginate in a concentration of from 0.5 to 2 wt. %, from 0.75 to 1.5 wt. % or 0.8 to 1 .2 wt. %. In some embodiments, each of the first and second polymer in a concentration of from 0.5 to 2 wt. %, from 0.75 to 1.5 wt. % or 0.8 to 1.2 wt. %. In some embodiments, the alginate in the mixture is provided at a weight ratio of alginate to a total of the first and the second polymer being of 1 :1.1 to 1.1 to 1 , 1 :1.05 to 1.05 to 1 or about 1 :1 (±5%). In some embodiments, the first polymer and the second polymer are provided in a weight ratio of 1 :1.1 to 1.1 :1 , 1 :1.05 to 1.05 to 1 or about 1 :1 (±5%).

[0131] In some embodiments, the hydrogel patches can be coated with any of the suitable polymers described above such as PADS or PHMS. The coating step can include contacting the hydrogel patch to a solution or suspension comprising 0.1 to 5, 0.2 to 3 or 0.5 to 1 wt. % of coating polymers.

[0132] The intersection of the string can form an infill pattern that can be a rectilinear, hexagonal, or triangular grid. There may also be a perimeter on each layer made out of the same material (with the same cells) or a material to impart greater mechanical strength (with or without cells). There may be a perimeter on every layer, every other layer, selected layers or the patch can be made with no perimeters, with the strings and filaments forming a self-supported structure. The overall patch shape can be a rectangular prism, but, preferably, the edges are rounded. The layers themselves may also be tapered in the height of the patch as well.

[0133] It should be noted that the hydrogel patches may shrink up to approximately 25% during gelation, and the degree of shrinking needs to be taken into account within the design.

[0134] Printing technologies offer multiple ways to improve the function of the printed patches. For example, multi-layer patches can be printed into shapes that facilitate rolling the patch along one axis, facilitating laparoscopic administration to the desired implantation site followed by unrolling. The hydrogel patches can be given dome-shaped structures by stepping the z- dimension upwards towards the interior of the hydrogel patch, effectively turning it into a flat dome that is much more resistant to folding deformations and other distortions. Some designs may facilitate implantation. For example, if printed with a 3,- 4- or, preferably, 6-axis printhead, the structure can have a curved, dome or bowl-like structure that may fit better into a specific intended implantation site, as well as further supporting the structure and limiting the chance of the patchdeforming during handling, implantation or long-term in vivo implantations. Further, the patch can be designed and sized for implantation into specific animal models, including in a rat intraperitoneal cavity, within fascia outside of the peritoneal cavity, or subcutaneously.

[0135] The printed hydrogel patch, after all post-processing steps, can be placed in a mould slightly larger than the printed structure and the encompassing hydrogel can be subsequently formed around it in the mould. The encompassing hydrogel may optionally contain (potentially recombinant human) collagen I, collagen III, collagen IV, laminin, fibrin, hyaluronic acid, etc., and combinations thereof. This mould may contain an array of pins, to reserve space for improved post-implant penetration of vasculature into the hydrogel patch.

[0136] The hydrogel patches described herein can be used for cellular therapy. For example, when the live cells are pancreatic islets or cell aggregates containing beta cells, the hydrogel patches can be implanted in the peritoneal cavity of a subject in need thereof for a period of at least 1 week, at least 2 weeks, at least one month, at least two months, at least three months, at least 6 months or at least 1 year. The implantation can also be made subcutaneously or specifically in the intraperitoneal cavity. The live cells remain alive throughout and also retain their cellular function (such as secreting insulin in the case of beta cells). An advantage of the hydrogel patch is that it can be easily explanted as it remains in a single form. As evidenced by the Examples, the cells can retain viability and cellular function through the manufacturing, implantation and explantation process.EXAMPLESynthesis of PM M- homocysteine thiolactone (PMM-HTL)

[0137] PMMAn (1 g, 6.41 mmol) was dissolved into 20 mL of acetonitrile in a 100 mL round bottom flask equipped with a magnetic stir-bar stirring at 700 rpm. To the stirring reaction mixture, triethylamine (TEA) (0.36 mL, 2.59 mmol) was added. Homocysteine thiolactone (197 mg, 1.28 mmol) that was dissolved in 4 mL of a 1 :1 v / v solvent mixture of dimethylsulfoxide (DMSO):acetonitrile (see Scheme 3 below). Once fully solubilized, the homocysteine thiolactone solution was added dropwise to the reaction mixture over 1-2 min. The reaction mixture was left overnight at room temperature (22 °C) and subsequently transferred into 12-14 kDa molecular weight cut-off (MWCO) cellulose acetate dialysis tubing and dialyzed against 1 % sodium chloride in distilled water for 2 days with twice daily changes, followed by one day of distilled water bath changed twice daily. It should be noted that the pH of the dialysis water bath and polymer solutionare inherently at pH 4-4.5, and hydrolysis of the thiolactone groups are minimized during purification at this pH. The dialyzed polymer solution was freeze-dried, resulting in PMM-HTL20 as a solid, obtained in 80-85 % yield.1H NMR spectrum of PMM-HTL was recorded in D2O on a Bruker™ Advance Neo 600 MHz spectrometer (Fig. 1).Scheme 3 dl-homocysteine thiolactone (20 moF TEA. 2 mol eq.acetonitrile, r t. o / n dl-homocysteinePMMAn thiolactoneSynthesis of PMM-CVS

[0138] The synthesis of PMM-CVS was performed similarly as previously described above for PMM-HTL by replacing HTL with CVS.Fluorescent Labeling of PMM-CVS and PMM-HTL Polymers

[0139] The fluorescent labelling of PMM-CVS and PMM-HTL was performed in a similar manner, using the following general procedure. PMMAn (1 g, 6.41 mmol) was dissolved in 20 mL of acetonitrile and transferred to a 100 mL round bottom flask equipped with a magnetic stir bar. Triethylamine (0.36 mL, 2.59 mmol), followed by fluorescein cadaverine (18 mg), were added to the reaction mixture stirring at 700 rpm at room temperature. The reaction mixture was left to react for 2 h before further post-functionalization reactions with either CVS or homocysteine thiolactone as described above.Synthesis of PADS Copolymers (poly[APM-co-DMAEA-co-SBMA])

[0140] PADS copolymers of various compositions were synthesized by reversible additionfragmentation chain-transfer (RAFT) polymerization, targeting molecular weights (MWs) from 5,000 to 50,000 g / mol at about 80 % total monomer conversion. To prepare PADS at a 1 :1 :2 APM:DMAEA:SBMA molar feed ratio at 15,000 g / mol, the following procedure was used. APM (0.406 g, 2.27 mmol) was dissolved in 4.4 mL of distilled water with 1.15 mol eq of HCI added with reference to the mol of DMAEA. DMAEA (0.325 g, 2.27 mmol) was added to the reactionmixture that was cooled in an ice-water bath, followed by pH adjustment to pH 3-4. SBMA (1.27 g, 4.55 mmol) was added to the reaction mixture, followed by a solution of 4-cyano-4- (phenylcarbonothioylthio)pentanoic acid (CTP) (30 mg, 0.108 mmol) and 4,4’-azobis(4- cyanovaleric acid) (V501) (6 mg, 0.021 mmol) in 2.2 mL of 1,4-dioxane, for a 2:1 water: 1 ,4- dioxane v / v ratio as the polymerization solvent mixture. The reaction vessel was equipped with a magnetic stir bar, sealed with a rubber septum, and purged with N2 at room temperature while stirring for 1 h. The reaction mixture was then placed into a 70 °C oil bath for 2 h, followed by quenching in an ice-water bath and exposure to air. Aliquots were sampled for1H NMR analysis in D2O to measure conversion. The polymerization mixture was transferred to 3.5 kDa MWCO dialysis tubing and was dialyzed against distilled water at pH 3 for a total of 2 days with twice daily water bath changes, using UV-vis to measure the absorbance of the dialysate to confirm no monomer or other small molecules were eluting from the polymer mixture. The polymer solution was freeze dried, resulting in PADS-112-15k as a light pink solid in 77 % yield.1H NMR analysis showed 89 % monomer conversion, and a copolymer composition of 23 % APM, 20 % DMAEA, and 57 % SBMA based on monomer consumption.1H NMR spectrum of PADS-112-15k was recorded in D2O on a Bruker Avance Neo 600 MHz spectrometer (Fig. 2).Fluorescent Labeling of PADS Copolymers

[0141] PADS copolymers were fluorescently labelled using either fluorescein isothiocyanate (FITC) or rhodamine B isothiocyanate (RBITC) using the following protocol. To label PADS-112- 15k with RBITC at a targeted degree of 0.5 mol% (relative to total monomer units), the PADS copolymer (150 mg) was first dissolved in 15 mL of distilled water in a 20 mL vial equipped with a stir bar. The pH was adjusted to pH 7.5 followed by the addition of RBITC (180 pL of 1 % solution in dimethyl formamide (DMF), 3.4 pmol) to the vigorously stirring reaction mixture. After 30 min, the reaction mixture was pH adjusted to pH 3-4 and transferred to 3.5 kDa MWCO dialysis tubing for purification as similarly described above. The polymer was then freeze dried, resulting in PADS-112-15k-RBITC as a magenta solid in about 80-85% yield.Synthesis of Homocysteine Thiolactone Based Copolymers

[0142] Homocysteine thiolactone methacrylamide was copolymerized to generate thiolactone reactive polymers for coating hydrogels anchored by covalent crosslinking with polyamines. As an example, homocysteine thiolactone methacrylamide (HTLMA) was copolymerized withmethacrylic acid and SBMA to form poly[HTLMA-co-methacrylic acid-co-SBMA] (PHMS) copolymers.

[0143] PHMS copolymers of varying compositions were synthesized by reversible-addition- fragmentation chain-transfer polymerization (RAFT) polymerization targeting molecular weights ranging from 5,000 to 50,000 g / mol at about 80 % total monomer conversion. To prepare PHMS with a targeted molar feed ratio of 1 :1 :3 for HTLMA:MAA:SBMA at a targeted MWof 25,000 g / mol, called PHMS-113-25k, the procedure was similar to the RAFT polymerization as described above to PADS copolymers. PHMS-113-25k by RAFT polymerization reached 91 % total monomer conversion after 2.5 h at 70 °C.Preparation of Thiolactone-Vinyl Sulfone Hydrogel Capsules

[0144] Gel forming solutions were prepared by combining PMM-CVS and PMM-HTL at 1 to 5 wt.% loadings with sodium alginate (NovaMatrix™, MVG) at 0.5 to 3 wt.% in 4-(2-hydroxyethyl)- 1 -piperazineethanesulfonic acid (HEPES) buffered saline (HBS). Sodium alginate was used partly as a gel forming processing aid to rapidly form an initial gel scaffold upon exposure to a gelling bath containing multi-valent cations such as calcium, barium, strontium, zinc, and iron. To prepare the core hydrogel capsules at 4 % total PMM-CVS / HTL and 1 % alginate at about 1000 pm in diameter, the following procedure was used. PMM-CVS (40 mg) and PMM-HTL (40 mg) were dissolved in 1 mL of HBS, adjusted to pH 7.2-7.7. To the polymer solution, 1 mL of 2 % sodium alginate in HBS was added, and the mixture was vortexed for 1-2 min, followed by filtering through a 0.22 pm syringe filter. The gel forming solution was then loaded into a 3 mL syringe and placed into a vertically oriented syringe pump (Harvard Apparatus), attached with a coaxial needle (Rame-Hart) with an 20G inner needle and 14G outer needle. The polymer solution was extruded into a calcium chloride gelling bath with liquid flow rate set to 15 mL / h and the coaxial air flow rate to 2.65 LPM. The hydrogel capsules were left to cure under stirring in the gelling bath for another 15 min, followed by washing steps with saline, and / or HBS solutions containing 0 to 100 mM calcium chloride and / or barium chloride, prior to subsequent coating steps.Coating of Hydrogel Capsules with Permeability Reducing MembraneSingle Coating (Fig. 4, Configuration 1)

[0145] Hydrogel capsules were coated with polycations containing primary amino groups to electrostatically form covalently crosslinked permeability reducing (size-exclusion) membranes.The amino groups of the polycation subsequently react with the thiolactone groups of the polymer in the hydrogel capsule or polymer coating to form a covalently linked network (described in greater details below).

[0146] To form PADS coated hydrogels with thiolactone groups in the core hydrogel capsule, the following procedure was used as a representative example. Thiolactone containing hydrogel capsules (4 wt.% PMM-CVS / HTL polymers, 1% alginate), in form of a ca 1.5 mL dense suspension of capsules as formed as described above, were exposed to 4.5 mL a solution of 1 wt.% PADS-112-15k in HBS containing 100 mM calcium chloride for 6 min with occasional swirling. The capsules were then left to settle and the supernatant was removed. The capsules were washed twice with saline and stored in saline for approximately 2 h at room temperature, for covalent crosslinking by aminolysis of thiolactone groups and subsequent thiol / ene reaction of the generated homocysteine thiols with vinylsulfone groups (Scheme 4).Scheme 4Ring openin by aminolysis R-N HHBS. pH 7 4. rt mm • 1 -2 hoursPMM-homocysteineDouble Coating (Fig. 4, Configuration 3)

[0147] To form hydrogel capsules with thiolactone groups in both the core hydrogel and coating of the capsule, the following was used as a representative example. Thiolactone containing hydrogel capsules (2 wt. % total PMM-CVS / HTL polymers, 1 wt.% alginate) formed as described above, were exposed to a solution of 0.5 wt.% PAD75-25k in HBS containing 50 mM of CaCh for 6 min with occasional swirling. The capsules were left to settle and supernatant was removed, and the capsules were washed twice with HBS containing 20 mM CaCh. The capsules were then exposed to 0.5 wt.% PHMS-60-25k in HBS containing 20 mM CaCh for 6 min with occasional swirling. The capsules were then washed twice with HBS and stored in HBS for approximately 2 hours at room temperature to allow for covalent crosslinking by aminolysis of thiolactone groups and subsequent thiol / ene reaction of homocysteine thiols with polymer-bound vinylsulfone groups.Preparation of Hydrogel Patches (Hydrogel patches)

[0148] To prepare hydrogel patches, the gel forming solution was prepared similarly to the solution for hydrogel capsules, with a representative example described as follows. PMM- CVS / HTL (50 mg each) were dissolved in 0.83 mL of HBS and combined with 4.17 mL of 2.4 wt.% alginate (NovaMatrix™, MVG), for a final concentration of 2 % total PMM-CVS / HTL and 2 % alginate, pH adjusted to pH 7.2-7.5. The gel forming solution was filtered through a 0.22 urn syringe filter and loaded into a 3 mL syringe.

[0149] The gel forming solution was used to print a 15 mm x 15 mm hydrogel patches patch using a BioX Bioprinter from Cellink™. These patches were generated at room temperature in a 6-well plate, with ~2 mL of LifeSupport™ printing bath in each well, using a rectilinear pattern with a 30% infill density, where the polymer was extruded at 4.25 pL / s and the print head speed was 10 mm / s. 2-3 mL of 12.5 mM calcium gelling bath was added to the well containing the construct and left to cure for 5 min. Following the calcium gelation step, the LifeSupport™ support bath was liquefied by placing the plate in a 37°C hybridizer oven for 30 min. The hydrogel construct was removed and placed into a well containing 3 mL of a 10 mM barium chloride gelling bath for 5 min, followed by 2 washes with 3 mL of saline and stored in saline prior to coatings.Coating of Hydrogel PatchesSingle Coating

[0150] Hydrogel constructs containing thiolactone groups were coated with polycations to form permeability reducing membranes, similar to hydrogel capsules and strings. A representative example of coating a hydrogel patch is provided as follows and shown in Fig. 3. Following formation, as described above, the hydrogel construct was placed into a 6-well plate and washed twice with 3 mL of HBS containing 50 mM calcium chloride. Once the supernatant was removed from the patch, 3 mL of 1 % PADS-112-25k (fluorescently labelled with rhodamine B) was added to the well and the plate was placed on an orbital shaker for gentle agitation (39cannin. 20-25 rpm) for 6 min. The supernatant was removed, and the coated hydrogel patch was washed 3 times with 3 mL of saline, and subsequently cured in saline for 2 h to allow for covalent crosslinking via aminolysis of thiolactone groups and thiolene reaction with homocysteine free- thiols with vinylsulfone groups.Double Coating

[0151] The hydrogel patches were transferred into a solution of the polycation PAD-75-25K at 1 wt. / v% in HBS for 3 min. The incubation step was performed on an orbital shaker with medium to high-speed. The polycation coated hydrogel patch was transferred to HBS solution for washing for 3 min, followed by exposure to HTL functional polyanion PHMS-60 at 1 wt. / v% in HBS for 6 min on an orbital shaker at med-high speed. The doubly coated hydrogel patch was then washed with HBS for 3 min, followed by incubation in HBS for approximately 2 h to allow for covalent crosslinking of the hydrogel core and coatings.Chemical Robustness Tests

[0152] To assess the coated hydrogels for covalent crosslinking, the hydrogel capsules, strings, and patches were subject to chelation of calcium using sodium citrate followed by an increase in pH to about pH 12 using 1 M NaOH to disrupt electrostatic complexation. Following this citrate-NaOH treatment, the samples were assessed by microscopy to determine if the hydrogels remained intact, or partially intact, which confirmed the presence of covalent crosslinkages.

[0153] As a representative example, the procedure for a citrate-NaOH test on coated hydrogel capsules is provided as follows. An aliquot (0.25 mL) of hydrogel capsule suspension with a 3:1 capsules to saline ratio, was added to a 4 mL glass vial, followed by 2 mL of 70 mM sodium citrate. The suspension was incubated with occasional mixing for at least 1 h before imaging for survival analysis.Microscopy

[0154] Brightfield and fluorescence images were taken with a Nikon T / Eclipse inverted microscope. Confocal laser scanning microscopy images were taken with a Nikon A1 Confocal T / Eclipse microscope.Membrane Permeability Studies

[0155] To test the permeability of the polymer membranes formed, the coated hydrogels were incubated with fluorescently labelled IgG and dextrans of various molecular weights. The procedure for the in-diffusion of IgG with hydrogel capsule formulations is described as follows. To a well in a glass-bottom 96-well plate, approximately 10-15 capsules were transferred, and the supernatant was removed. To the capsules, 150 pL of 500 pg / mL solution of lgG-alexafluor488 in HBS was added, followed by pipette aspiration for mixing of the supernatant. The capsuleswere imaged by confocal laser scanning microscopy immediately after sample preparation, and at various time intervals thereafter.

[0156] In-diffusion was measured by selecting multiple circular regions of interest (ROIs) from both within the capsules, and outside the capsules. The mean fluorescence intensity in the FITC channel of the ROIs inside the capsule was divided by the corresponding intensity of the ROIs outside of the capsule to obtain the in-diffusion ratio.Results and Discussion:

[0157] As discussed above, cell therapies such as type 1 diabetes therapies involve transplantation of cells. In the case of type 1 diabetes, the encapsulated cells are beta cells and they require exclusion of auto-antibodies IgG in order to stop the auto-immune response from eliminating the newly transplanted cells as well. The present hydrogels fulfil the need to more tightly control the permeability of the resulting encapsulating hydrogel in order to exclude at least some of the larger auto antibodies known to pose specific dangers to transplanted beta cells.Formation of Coated Hydrogels with Permeability Reducing Membrane

[0158] Coated hydrogels with covalent crosslinking based on thiolactone chemistry were prepared with reactive groups used in the configurations shown in Fig. 4.

[0159] Hydrogel capsules containing homocysteine thiolactone groups in the core were prepared using by using PMM-HTL and PMM-CVS at varying total wt.% loadings along with 1 wt.% alginate in the gel forming solutions (Fig. 4 Configuration 1).

[0160] As an example, 2 % PMM-CVS / HTL (1:1 wt ratio) and 1 % alginate hydrogel capsules were coated with 0.3 wt.% PADS-112-15k, called formulation A. (Fig. 5A). Formulation A was used to encapsulate human donor islets (Fig. 5B). The islets within A appeared to survive the encapsulation and coating processes, suggesting promising islet and cell compatibility with the thiolactone-polyamine based approach.

[0161] Formulation A was prepared with fluorescent derivatives of the polymers, using PMM- CVS-FITC (fluorescein) and PADS-112-15k-RBITC (rhodamine B). The capsules were imaged by confocal laser scanning microscopy and cross-sectional images were obtained to observe the distribution of the polymer components within the hydrogel network (Figs. 6A-6C).

[0162] Formulation A appeared to have a relatively homogeneous core consisting of PMM- CVS as visualized in the FITC channel (Fig. 6A). PADS-112-15k appeared to form a polymer membrane, with higher PADS concentration at the surface of the hydrogel and some diffused throughout the core. It is anticipated that the polymer within the core of the hydrogel may not be as cyto-toxic as polycations in solution state as it is complexed with the polyanionic hydrogel of PMM-CVS / HTL and calcium alginate, thus de-risking the cytotoxicity of the polymer within the hydrogel. The permeability of the membrane of formulation A was assessed and presented further below.

[0163] Formulation A hydrogel capsules were subjected to an aggressive chemical stress test using sodium citrate and sodium hydroxide to probe the presence of covalent crosslinking of the hydrogel network (Figs. 7A-7C). Formulation A survived treatment with sodium citrate, which chelated the calcium from the hydrogel core containing calcium alginate. The capsules were able to swell and withstand the osmotic stress during that process. Following citrate treatment, 1 M sodium hydroxide was added to the sample to increase the pH to approximately 12 to disrupt the electrostatic interactions of the PADS copolymer with the hydrogel core by deprotonating the ammonium cations. Some capsules appeared to ruptured after the addition of NaOH, however the capsule membrane appeared to have remained intact, which suggests the that PADS copolymer had indeed reacted with homocysteine thiolactone groups of PMM-HTL in the core hydrogel capsule. These results are demonstrate that covalently crosslinked hydrogels with thiolactone-polyamine chemistry are a viable platform to generate permeability controlling membranes for hydrogels.

[0164] In another experiment, hydrogel capsules were prepared using 4 wt.% total PMM- CVS / HTL (1 :1 wt. ratio), 1 % alginate, and coated with 1 wt.% PADS-112-15k, called formulation B (Fig. 8), which was also used to encapsulate human donor islets (Fig. 9).

[0165] Brightfield images of formulation B appeared much more opaque relative to A, due to the higher polymer loading of the PMM-CVS / HTL polymer in the hydrogel gel network with alginate and likely nano-scale phase-separation of the polymers at these higher concentrations. This phase separation of the PMM-CVS / HTL polymer within the calcium alginate core is thought to aid in covalent crosslinking by increasing the local concentration of the PMM polymers and thus increasing the rate of reaction between free-thiols of ring-opened thiolactone groups and vinyl sulfone groups.

[0166] In yet another experiment, coated hydrogel capsules were made with 2 % total PMM- CVS / HTL (1 :1 wt. ratio), 1 % alginate, and were coated with 1 % PADS-112-25k (Fig. 10). These higher molecular weight PADS polyamines (25k, versus 15k as used in A and B) were anticipated to be more restricted to the surface of the hydrogel during the coating process, forming shell membranes that may be thinner, but denser, thus further decreasing shell permeability.

[0167] The permeability of coated hydrogel capsule formulations A, B, and C were tested using fluorescently labelled IgG. The permeability of the coated capsules were compared to uncoated capsule formulation blank. As a representative example, a confocal cross-sectional image of C is shown after 24 h of incubation with lgG-alexafluor488 in Figs. 11A-11 B. The indiffusion of IgG was analyzed by measuring the fluorescence intensity of IgG within the capsule and dividing that value by the fluorescence intensity in the supernatant, using representative regions of interest (ROIs) as described above. The results for this analysis of the capsules incubated with IgG for about 24 h were compiled in Fig. 12.

[0168] The data in Fig. 12 shows the progressive exclusion of IgG due to the membranes formed from the polymer coating formulations based on thiolactone-polyamine crosslinking chemistry. This demonstrates the ability to reduce the permeability and thus increase the exclusion of 150 kDa antibodies, using the thiolactone-polyamine crosslinking chemistry.

[0169] It is common in the hydrogel science to use permeability of defined molecular weight probes, such as fluorescently labelled dextrans or IgG, to measure pore dimensions under physiological conditions, e.g. in hydrated state and at pH 7.4 in saline. Dextrans and IgG of 150,000 kDa are known to have hydrodynamic diameters of 18 nm and 12.7 nm, respectively, and the almost complete exclusion of these probes from the capsule interior after 24 hrs sets an upper diameter limit of 12.7 nm for the pore dimensions of the capsule shells.

[0170] In a further experiment, coated hydrogel capsules were prepared with thiolactone containing polymers as part of a doubly coated capsule formulation. The doubly coated capsule formulation may be beneficial as adding a final layer to the permeability controlled membrane may be used to improve the anti-fibrotic properties of the surface of the capsules. Formulation D was coated with PHMS-60-25k that was fluorescently labelled with rhodamine B. Confocal fluorescence imaging of D in the TRITC channel showed a distinct layer on the surface of the capsule (Fig. 13).

[0171] In another experiment, doubly coated capsule formulation was prepared with higher a concentration of PAD75-25k and PHMS-60-25k at 0.75 wt. / v% in each coating step, and was labeled as formulation E. Human donor islets were encapsulated in E doubly coated capsules at 3 and 6 lEQ / capsule densities along with a blank, non-islet containing control. The permeability of the capsules was tested with IgG after 24 h incubation and showed low (between 20-30%) diffusion for blank and both low and high islet containing capsule densities (Fig. 14).

[0172] In another experiment, rat islets were encapsulated in formulation D hydrogel capsules and implanted into diabetic rats. After 2 weeks in vivo, the capsules were explanted to test the anti-fibrotic properties of the PHMS-60-25k polymer on the surface of the doubly coated hydrogel. The retrieved capsules were imaged on a Nikon™ Inverted Ti microscope with the brightfield channel and a large stitched image was taken to capture the entirety of the sample (Fig. 15). The retrieved D capsules containing rat islets appeared intact and showed little to no cellular overgrowth. These results demonstrate that the doubly coated capsule formulation with PHMS- 60 on the surface of the hydrogel was successful in preventing a host-response and fibrosis of the implanted material.Hydrogel Patches

[0173] In another experiment, the gel forming polymers of the hydrogel capsule formulations were used to demonstrate the translation of the thiolactone-polyamine-thiolene crosslinking chemistry to other hydrogel shapes. PMM-CVS / HTL (2 wt.%) and alginate (2 wt.%) were combined and extruded using a 3D bioprinter to form a hydrogel construct herein called a hydrogel patch. The thiolactone containing hydrogel patch was prepared, forming a 15 x 15 mm rectilinear pattern, 3-layers thick. The patch was coated with PADS-112-25k (1 wt.%, rhodamine B labeled), forming coated patch formulation P-001. Overview stitched images of P-001 were captured by brightfield (Fig. 16A) and fluorescence microscopy (Fig. 16B).

[0174] Patch P-001 was imaged by confocal laser scanning microscopy to visualize the distribution of the PADS polymer coating onto the hydrogel patch filaments (Fig. 17A). Since the patch was printed in a support bath, the filaments appear rough on the surface due to the imprinting of the support bath particles onto the hydrogel (Fig. 17B). Regardless, the PADS copolymer appeared to coat the filaments of the patch, with greater density at the surface and limited diffusion into the hydrogel.

[0175] The hydrogel patch P-001 was treated with sodium citrate followed by sodium hydroxide to test whether the coated patch was covalently crosslinked (Figs. 18A-18B). The filaments of the hydrogel patch coating appeared to survive this chemical stress test, as seen by the remaining fluorescence of the patch.Rat-sized model 3-layer hydrogel patches

[0176] A 1.4 by 1.2 cm 3-layer hydrogel patch was produced for rat model studies without immune suppression. The gel formers (1 wt% of each PMM-CVS20 and PMM-HTL20) were dissolved in HBS along with 2.5 wt% alginate. The pH of this solution was adjusted to -7.2-7.6. LifeSupport™ supporting bath was prepared as described in its instructions, using 20 mM CaCh as the solvent, and left in the fridge until printing occurred. Immediately before the print, -20,000 I EQ of primary human islets were added to 0.7 mL of the gel former solution and the mixture was placed in a syringe and attached to the printhead for printing. The 1.4 by 1.2 cm 3-layer print design had a triangular infill pattern with a 34% infill density, and the perimeter around the structure was comprised of the same material as the infill.

[0177] The printing was performed with a 20G nozzle with a flow rate of 7 pL / s and a tip speed of 10 mm / s. Once the print was completed, a solution of 100 mM CaCh was added to the printing bath and the patch was left submerged for 15 minutes. Subsequently, the LifeSupport™ was removed by heating the print bed to 37°C for 5-30 minutes before imaging and rinsing with saline. After the saline rinse, the hydrogel patches were submerged in islet media and placed in the incubator for Live / Dead assays and Glucose Stimulated Insulin Secretion (GSIS) assessments. The Live / Dead assessment one day after printing is shown in Figs. 19A-19C. This highlights the fact the cells are healthy after the printing process. The GSIS results, show in Figs. 20A-20B, indicate good islet functionality in response to glucose stimulation after 3 and 5 days of being encapsulated into hydrogel patch systems with varying degrees of alginate content.Doubly coated 1.5 by 1.53-layer hydrogel patch

[0178] In order to limit the diffusion of compounds that could promote the immune response, including exosomes, proteins, etc., while allowing for diffusion of oxygen and insulin, a double coating process was employed to tune the molecular weight cut-off of these 3D bioprinted structures. The double coating process itself involves the following steps:(i) Transfer the bioprinted hydrogel patches into a solution of the first coating polymer (the concentration of the coating polymer can be between 0.1 and 5 wt. / v% (preferably between 0.5 and 1.0 wt. / v%). The coating polymer can be made of different materials, such as PAD-25-25K. The coating polymer exposure time and CaCh concentration of the HBS buffer used for dissolving it can vary based on the required exclusion percentage. The incubation time can be from a few seconds to 30 mins, preferably between 3 and 6 mins. The CaCh concentration of the HBS buffer used for coating polymer solution can vary from 0 to 100 mM, preferably between 0 and 50 mM, based on the required exclusion percentage. The incubation step can be performed on an orbital shaker with medium to high-speed.(ii) Transfer the coated hydrogel patches to the HBS solution for washing. The incubation time can vary from a second to 5 mins. This can affect the coating polymer thickness and exclusion percentage.(iii) Transfer the coated hydrogel patches into a solution of the first coating polymer (the concentration of the coating polymer can be between 0.1 and 5 wt. / v% (preferably between 0.5 and 1.0 wt. / v%)). Based on our coating strategy, the coating polymer can be made of different materials, such as PH MS-60. The coating polymer exposure time and CaCh concentration of the HBS buffer used for dissolving it can vary based on the required exclusion percentage. The incubation time can be from a few seconds to 30 mins, preferably between 3 and 6 mins. The CaCh concentration of the HBS buffer used for coating polymer solution can vary from 0 to 100 mM, preferably between 0 and 50 mM, based on the required exclusion percentage. The incubation step can be performed on an orbital shaker with medium to high-speed(iv) Let the coated hydrogel patches stay in HBS, or islet supporting media including serum and plasma for 2 hrs to crosslink fully.(v) Transfer the coated hydrogel patches to the 0.1 wt% Dextran-Fluorescein 70k solution and let the Rafts incubate there overnight (24hrs). The 0.1 wt% Dextran-Fluorescein 70k volume was 1.5 mL in a 24-well plate.

[0179] The concentration of the coating polymers, PAD-25-25K and PHMS-60, was 1.0 wt. / v% for the latest experiment. The incubation time of the hydrogel patch with coating polymers was 6 min. The HBS washing time was 3 mins. As can be seen in Fig. 21 , the double-coatingstrategy successfully reduced the Dextran-Fluorescein 70K’s in-diffusion percentage from approximately 46% (for non-coated hydrogel patch) to 24-25% (for the coated hydrogel patches) after 24 hours. Representative images that lead to these results are shown in Figs. 22A-22C and 23A-23C.Cell-free Human / porcine prototype

[0180] A large 3-layer hydrogel patch with a size of 4.5 cm by 4 cm, which would be suitable for porcine in vivo studies, is shown in Fig. 24. It was printed by using the gel formers (1 wt% of each PMM-CVS20 and PMM-HTL20) that were dissolved in HBS along with 2.0 wt% alginate. The hydrogel patch was printed in LifeSupport™ supporting bath prepared as described in its instructions, using 12 mM CaCh as the solvent, and left in the fridge until printing occurred.

[0181] The 4.5 by 4.0 cm 3-layer print design had a triangular infill pattern with a 34% infill density, and the perimeter around the structure was comprised of the same material as the infill. The printing was performed with a 20G nozzle with a flow rate of 7.0 pL / s and a tip speed of 14 mm / s. Once the print was completed, a solution of 100 mM CaCh was added to the printing bath and the patch was left submerged for 15 minutes. Subsequently, the LifeSupport™ was removed by heating the print bed to 37°C for 5-30 minutes before imaging and rinsing with saline. After the saline rinse, the hydrogel patches were submerged in islet media and placed in the incubator for Live / Dead and GSIS assessments.

[0182] After successful extrusion (i.e. the size of the filaments are similar to the inner diameter of the nozzle), this particular design has a volume of ~1.0 mL. This means that hydrogel patches of this size could be easily adapted to possess an islet encapsulation amount of 10,000 - 75,000 lEQ / patch.Vascularization of hydrogel patch

[0183] A 3-layer cell-free 1.4 x 1.2 cm patch with rounded edges was printed where the interior of the patch included 1 wt.% of each of our PMM-CVS20 and PMM-HTL20 polymers along with 2 wt% alginate. The perimeter of the patch comprised 3 wt% alginate to provide greater mechanical properties. The printing was performed in a bath of LifeSupport™ particles with 12.5 mM CaCh and the infill density of the triangular patch design was 34%. The print itself was performed with 20G needles for each component.

[0184] Once the print was completed, the patch was left submerged with 25mM CaCl2 for 15 minutes, the LifeSupport™ was removed in a 37°C incubator for 45 minutes before imaging (Fig. 25A) and rinsing. After this, the patch was implanted into the pre-intraperitoneal space of a rat and left for 4 weeks. After 4 weeks, the patch was explanted and showed evidence of vascular ingrowth (Fig. 25B). The degree of vascular ingrowth may be enhanced by adjusting the infill density of the patch or including pro-angiogenic factors or cells in the material.Conformal coating

[0185] Islets were directly coated with PAD75-25k and PHMS-60 to form covalently crosslinked conformal coatings. As a representative example, 20-30 rat islets were transferred into a 5 mL microtube and washed twice with HBS. The islets were left to settle and the supernatant was removed from the islets. To the islets, 1 mL of 0.5 wt.% of PAD75-25k in HBS was added and left to incubate at room temperature for 2 min, followed by washing with 1 mL of HBS twice to remove excess polycation in the supernatant. To the singly coated islets, 1 mL of 0.5 wt.% PHMS-60-25k (rhodamine labelled) in HBS was added to the islets and left to incubate for 2 min at room temperature, followed by washing with 1 mL of HBS twice. The doubly coated islets were placed in supplemented media and imaged on the confocal to visualize the PHMS-60- 25k (rhodamine labelled) coating on the islets in the TRITC channel.

[0186] In another experiment, rat islets were doubly coated with first PAD75-25k, followed by PHMS-60-25k to form covalently crosslinked conformal coatings. The coated islets were imaged with a Nikon A1 confocal microscope to visualize the rhodamine labelled PHMS-60-25k coating in the TRITC channel. A confocal cross-sectional images of a representative conformally coated islet is shown in a merged image of brightfield and TRITC channels (Fig. 26A) and TRITC channel (Fig. 26B). Figs. 26A-26B show a thin layer (approx. 10-15 urn) of PHMS-60-25k had indeed formed a conformal coating on rat islets while maintaining good morphology of the islets which also demonstrated good cell compatibility of the polymers.

Claims

WHAT IS CLAIMED IS:

1. A method of printing a hydrogel patch containing live cells, the method comprising: extruding a mixture comprising alginate, a first polymer, a second polymer, and live cells through a needle or nozzle, wherein the first polymer is functionalized with a homocysteine thiolactone of formula I, wherein any three of the R2 are H, and the remaining R2 is selected from H, C1-C4 branched or linear alkyl, methoxy, ethoxy, and halogen;wherein the second polymer is functionalized with a pendant vinyl group on a second polymer; while extruding, moving the needle or the nozzle to form a precursor of the hydrogel patch; gelling the alginate, preferably by submersion of the precursor of the hydrogel in a bath containing calcium, strontium, barium, iron, or zinc ions to gel the alginate; and reacting the first polymer functionalized with the homocysteine thiolactone of formula I to open the ring and obtain a pendant thiol group, and crosslinking the pendant thiol group of the first polymer with the pendant vinyl group on the second polymer to obtain the hydrogel patch containing live cells.

2. The method of claim 1 , wherein the mixture is extruded in layers, preferably wherein each layer has the same or substantially the same size and shape.

3. The method of claim 2, wherein each layer comprises a substantially planar array of intersecting strings defining apertures therebetween having a surface area of 100,000 to 500,000 pm2, wherein the hydrogel precursor comprises at least two layers.

4. The method of any one of claims 1 to 3, wherein the first polymer and / or the second polymer have a backbone selected from poly(methyl vinyl ether-alt-maleic anhydride) (PMM), homopolymer of methacrylic anhydride, homopolymer of acrylic acid, homopolymer of methacrylic acid, or copolymers of acrylic acid and methacrylic acid, or copolymers of maleic anhydride with vinyl ethers selected from methyl vinyl ether, ethyl vinyl ether, propyl vinyl ether, butyl vinyl ether, hydroxy ethyl vinyl ether, and methoxy ethyl vinyl ether, copolymers of N-vinylpyrrolidone with maleic or itaconic or citraconic anhydrides.

5. The method of any one of claims 1 to 4, wherein the first polymer and / or the second polymer has a backbone that comprises monomeric units selected from 2-aminoethyl acrylate, 2-aminoethyl methacrylate, 2-aminoethyl acrylamide, 2-aminoethyl methacrylamide, 3-aminopropyl acrylate, 3-aminopropyl methacrylate, 4-aminobutyl acrylate, 4-aminobutyl methacrylate, 4-aminobutyl acrylamide, 4-aminobutyl methacrylamide, acrylic acid (AA), acrylamide, methacrylic acid (MAA), 2- hydroxypropylacrylamide (HPA), 2-hydroxypropylmethacrylamide (HPMA), 2- hydroxyethylacrylate (HEA), 2-hydroxyethylmethacrylate (HEMA), 2-(methacryloyloxy)ethyl 2-(trimethylammonio)ethyl phosphate (MPC), carboxybetaine methacrylate (CBM), carboxy betaine methacrylamide, N,N-dimethylaminoethylacrylate(DMAEA), N,N-dimethylaminoethyl methacrylate, 3-N,N-dimethylaminopropyl acrylamide, 3-N,N-dimethylaminopropyl methacrylamide, 3-N,N-dimethyliaminopropyl acrylate, 3- N,N-diaminopropyl methacrylate, 4-N,N-dimethylaminobutyl acrylamide, 4-N,N- dimethylaminobutyl methacrylamide, 1,3-bis(dimethylamino)propyl acrylate, 1,3- bis(dimethylamino)propyl methacrylate, 2-((2-(dimethylamino)ethyl)(methyl)amino)ethyl methacrylate, 2-((2-(dimethylamino)ethyl)(methyl)amino)ethyl acrylate, sodium acrylate, sodium methacrylate, sodium ethacrylate, hydroxyethyl acrylate, hydroxyethyl methacrylate, [2-(acryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, [2- (methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, 3-((3- methacrylamidopropyl)dimethylammonio)propane-1 -sulfonate, 2-(acryloyloxy)ethyl phosphate, 2-(methacryloyloxy)ethyl phosphate, (2-(methacryloyloxy)ethyl)phosphonate,(2-(acryloyloxy)ethyl)phosphonate, (2-acrylamidoethyl)phosphonate, (2-methacrylamidoethyl)phosphonate, 2-methacryloyloxyethyl phosphorylcholine, N- (carboxymethyl)-2-(acryloyloxy)-N,N-dimethylethan-1-aminium, N-(carboxymethyl)-2- (methacryloyloxy)-N,N-dimethylethan-1-aminium, N-(carboxymethyl)-3-methacrylamido-N,N-dimethylpropan-1-aminium, N-(carboxymethyl)-3-acrylamido-N,N-dimethylpropan-1- aminium, 2-(dimethylamino)ethyl acrylate, 2-(dimethylamino)ethyl methacrylate, / \ / -[3- (Dimethylamino)propyl] acrylamide, / V-[3-(dimethylamino)propyl] methacrylamide, / \ / -(3- Aminopropyl)acrylamide hydrochloride, / V-(3-aminopropyl)methacrylamide hydrochloride, vinyl sulfonic acid, vinyl phosphonic acid, 3-sulfopropyl methacrylate potassium salt, 2- (methacryloyloxy)ethanesulfonic acid, and combinations thereof.

6. The method of any one of claims 1 to 5, wherein the reacting step involves or is preceded by a hydrolysis or an aminolysis.

7. The method of any one of claims 1 to 6, wherein the needle or nozzle has a diameter of from 200 to 1 ,200 pm.

8. The method of any one of claims 1 to 7, wherein the mixture is an aqueous mixture comprising from 0.5 to 2 wt. % alginate, from 0.25 to 1 wt. % of the first polymer and fromO.25 to 1 wt. % of the second polymer.

9. The method of any one of claims 1 to 8, wherein the alginate in the mixture is provided at a weight ratio of alginate to a total of the first and the second polymer being of 1 :to 1.1 , to 1.1 to 1.

10. The method of any one of claims 1 to 9, wherein the first polymer and the second polymer are provided in a weight ratio of 1 : 1.1 to 1.1 : 1.

11. The method of any one of claims 1 to 10, wherein the bath comprises up to 100 mM of a total of the calcium, strontium, barium, iron, or zinc ions.

12. The method of any one of claims 1 to 11 , wherein the pendant vinyl group is a pendant vinylsulfone, and / or all of the R2 is H.

13. The method of any one of claims 1 to 12, further comprising coating the hydrogel with a third polymer such as poly[APM-co-DMAEA-co-SBMA] (PADS).

14. The method of any one of claims 1 to 13, wherein the live cells include cell aggregates, preferably pancreatic islets.

15. A hydrogel patch as obtained by the method of any one of claims 1 to 14.

16. A hydrogel patch comprising ionically crosslinked alginate; a polymer formed by the covalent crosslinking of by a first polymer that is functionalized with a homocysteine thiolactone of formula I, wherein any three of the R2 are H, and the remaining R2 is selected from H, C1-C4 branched or linear alkyl, methoxy, ethoxy, and halogenand a second polymer that is functionalized with a pendant vinyl group on a second polymer; and live cells encapsulated in the alginate and the polymer of the hydrogel patch.

17. The hydrogel patch of claim 16, wherein the hydrogel patch has layers as defined in claim 2 or 3, wherein the first and / or the second polymer are as defined in any one of claims 4, 5, 12 or 13; wherein the alginate and the first and second polymer are provided in amounts as defined in any one of claims 8 to 10; and / or wherein the live cells are as defined in claim 14.

18. The hydrogel patch of any one of claims 15 to 17, and incorporating a number of islet equivalents (I EQ) ranging from 1 ,000 to 100,000.

19. The hydrogel patch of any one of claims 15 to 18, wherein the hydrogel patch has a surface area of its layers being of from 1 cm2to 100 cm2.

20. Use of the hydrogel patch as defined in any one of claims 15 to 19, for promoting the production of insulin in an individual in need thereof.

21. Use of the hydrogel patch as defined in any one of claims 15 to 19, for treating diabetes.

22. A method of treating diabetes comprising transplanting in a subject in need thereof the hydrogel patch as defined in any one of claims 15 to 19.

23. The method of claim 22, further comprising explanting the hydrogel patch at least a week after transplanting in the subject.

24. The method of claim 23, wherein the live cells remain alive and are beta cells that produce insulin and still produce insulin when explanted.

25. The method of any one of claims 22 to 24, wherein the hydrogel patch is implanted in the peritoneal cavity.

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