Thiol-ene hydrogels

Abstract

A hydrogel string for encapsulating a biological material is provided. A hydrogel string has a thiol-ene cross-linked polymer having a first side chain functionalized backbone polymer functionalized with an activated olefin that is cross-linked with a free or protected thiol-containing group present on a second side chain functionalized backbone polymer. The biological material is encapsulated in a thiol-ene cross-linked polymer.

Description

[0001] Related Applications

[0002] This disclosure claims priority to U.S. Provisional Application No. 63 / 408,049, filed on September 19, 2022, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure relates to the field of hydrogel polymers, and in particular to encapsulating biological materials such as cells, cell aggregates, tissues, etc. within hydrogel polymers. Background Art

[0004] Synthetic polymers are chemically defined, scalable, and increasingly used to form hydrogels, often by employing efficient, biocompatible cross-linking chemistries such as "click reactions". Synthetic polymer hydrogels display many features reminiscent of the natural extracellular matrix (ECM) and are therefore being explored for use as ECM mimics. They can provide structural integrity to tissue constructs, control drug delivery, and serve as immunoisolation barriers for transplantation of therapeutic cells.

[0005] Typically, these reactive polymers are combined with an aqueous solution of therapeutic cells or model cells containing sodium alginate and dropped into a gelling bath containing calcium or barium to form calcium alginate or barium alginate beads containing one or two mutually reactive polymer gel formers. When only one reactive polymer is included in these alginate beads, a second gel former (sometimes referred to as a cross-linking agent) must be introduced into the beads by intradiffusion after bead formation.

[0006] Alginate chemistry has been thoroughly studied, but there is still much to be done for any alginate-based encapsulation material to be therapeutically successful in vivo. Thus, improvements in synthetic polymer chemistry and / or physical properties of combined hydrogels are needed. Summary of the invention

[0007] In one aspect, a hydrogel string is provided, comprising a thiol-ene cross-linked polymer, the polymer comprising a first side-chain functionalized main-chain polymer functionalized with an activated olefin (the olefin is cross-linked with a free or protected thiol-containing group present on a second side-chain functionalized main-chain polymer), and a biomaterial encapsulated in the thiol-ene cross-linked polymer. In this context, a hydrogel string refers to a string structure comprising at least one hydrogel polymer. In some embodiments, the hydrogel string has an aspect ratio of at least 5, preferably at least 100. In some embodiments, the hydrogel string comprises alginate, hyaluronic acid, gelatin, hydroxypropyl cellulose, carboxymethyl cellulose, methyl cellulose or elastin. In some embodiments, the biomaterial is a cell, a cell aggregate or a cell spheroid, and the hydrogel string optionally further encapsulates angiogenic agents and / or chemotactic agents. In some embodiments, the activated olefin is vinyl sulfone, maleimide, acrylate or methacrylate. In some embodiments, the thiol-containing group is 2-pyridinethiol or cystamine. In some embodiments, the backbone polymer is a homopolymer of polyacrylic acid, a homopolymer of polymethacrylic acid, or a copolymer of acrylic acid and methacrylic acid. In some embodiments, the hydrogel column further comprises a capping agent for neutralizing vinyl sulfone groups on the surface of the hydrogel column. In some embodiments, the hydrogel column has an outer diameter of less than 2000 μm, preferably less than 1000 μm, and more preferably less than 600 μm. In some embodiments, the thiol-ene cross-linked polymer forms an outer shell of the core encapsulating the biomaterial. In some embodiments, the shell has a non-uniform density, wherein the density of the outer surface as measured by fluorescence microscopy is higher than that of the inner surface.

[0008] In one aspect, a three-dimensional hydrogel structure formed by interconnected hydrogel columns as defined herein is provided, comprising a plurality of thiol-ene cross-linked polymers forming at least a portion of the interconnected hydrogel columns, wherein each hydrogel column is connected by thiol-ene cross-links forming a continuous cross-linked structure. In some embodiments, the three-dimensional hydrogel structure is a patch formed by 3D printing the hydrogel columns into a shape, wherein the hydrogel columns or portions thereof intersect or intersect with each other to form a two-dimensional array of micropores, preferably designed to maximize the surface area required for metabolic exchange of therapeutic cells, preferably wherein biomaterials can migrate between the intersecting columns or portions thereof.

[0009] In one aspect, a method for producing a hydrogel column is provided, comprising: continuously extruding or co-extruding a first polymer containing a free or protected thiol group and a second polymer containing a vinyl group into a bath containing a reactant, preferably a water bath, to drive gelation of the first polymer and the second polymer; and allowing a cross-linking reaction to occur between the thiol group and the vinyl group. In another aspect, a method for producing interconnected hydrogel columns is provided, comprising: extruding or co-extruding a composition comprising a first polymer containing a free or protected thiol group and a second polymer containing a vinyl group into a bath containing a reactant, preferably a water bath, to drive gelation of the first polymer and the second polymer, wherein the bath has a low reactant concentration, the low reactant concentration is a level capable of partially cross-linking the thiol-ene polymer to form a plurality of polymer columns; forming the plurality of polymer columns into a desired shape; and exposing the plurality of polymer columns to a reducing agent to further cross-link the plurality of polymer columns to form interconnected hydrogel columns. In some embodiments, extrusion or coextrusion also includes extruding or coextruding a biocompatible polymer with the first and second polymers, the biocompatible polymer selected from the group consisting of: alginate, hyaluronic acid, gelatin, hydroxypropyl cellulose, carboxymethyl cellulose, methyl cellulose or elastin. In some embodiments, the biocompatible polymer is alginate, and the bath contains ions selected from the group consisting of: calcium, barium, strontium, copper, zinc, manganese, cobalt, lead, iron or aluminum, preferably ions are present at a concentration of 5mM to 100mM. In some embodiments, the reactant is a reducing agent, preferably tris (2-carboxyethyl) phosphine (TCEP) or tris (hydroxypropyl) phosphine (THPP), and preferably the reactant is present at a concentration of 5mM to 100mM. In some embodiments, the method also includes providing a capping agent introduced as a final process step before the final wash to convert the residual vinyl sulfone group into a more biocompatible group. As used herein, "biocompatible polymer" refers to a polymer that does not induce an immune response in vivo, particularly when implanted in a mammal, it does not substantially interfere with the viability and function of the encapsulated cells. The biocompatible polymer may also be characterized by low fibrosis, such as a PCO score of 0-50%, preferably 0-25%, or more preferably 1-2%. In some embodiments, the ratio of thiol groups to vinyl (ene) groups is 0.95:1.05 to 0.65:1.35, preferably 0.9:1.1 to 0.5:1.5, and most preferably 0.8:1.2 to 0.6:1.4. In some embodiments, the method further comprises extruding or co-extruding the polymer into a bath through a needle (preferably a blunt-ended syringe). In some embodiments, the thiol group of the first polymer is a protected thiol group, preferably the first polymer is a poly (methyl vinyl ether-alternating-maleic anhydride) with a protected thiol group.

[0010] In one aspect, a graft is provided, comprising at least one hydrogel column as defined in any one of claims 1 to 11 and an optional supporting substrate, wherein at least one hydrogel column is substantially retrievable from the implantation site in a living mammal after a transplantation time of six weeks to one year or more (e.g., 2-3 years). As used herein, the term "substantially retrievable" means that the hydrogel column is retrieved, but when the retrieval occurs, a portion of the column may have degraded or decomposed. In addition, a portion of the column may remain in the body and cannot be retrieved, however, this portion is small and does not have any long-term effects on the subject receiving the graft. In one embodiment, at least 90% of the hydrogel column is retrieved. A method of implanting the described implantable device is also provided, comprising: depositing at least one hydrogel column containing a biomaterial onto omental tissue of a subject in need thereof, and folding and fixing the omental tissue by suturing or using tissue glue (preferably fibrinogen and / or thrombin glue). The method can be an open surgery or a laparoscopic procedure.

[0011] In another aspect, a hydrogel is provided, comprising a first side chain functionalized main chain polymer functionalized with an activated olefin (the olefin is cross-linked with a cystamine-containing group present on a second side chain functionalized main chain polymer). In some embodiments, the hydrogel further comprises alginate, hyaluronic acid, gelatin, hydroxypropyl cellulose, carboxymethyl cellulose, methyl cellulose or elastin. In some embodiments, the hydrogel encapsulates a biological material of mammalian or bacterial origin, preferably a cell, a cell aggregate or a cell sphere. In some embodiments, the activated olefin is vinyl sulfone, acrylate or methacrylate. In some embodiments, the main chain polymer is a homopolymer of polyacrylic acid, a homopolymer of polymethacrylic acid, or a copolymer of acrylic acid and methacrylic acid. In some embodiments, the cystamine-containing group has a poly(methyl vinyl ether-alternating-maleic anhydride) main chain polymer. In some embodiments, the hydrogel further comprises an end-capping agent for neutralizing vinyl sulfone groups on the surface of the hydrogel column. In some embodiments, the hydrogel is a capsule, a column or a patch. In some embodiments, the hydrogel is cross-linked.

[0012] In an additional aspect, a kit for encapsulating biomaterials (such as cells) is provided, comprising: a solution containing 5 mol.% to 35 mol.% of a polymer containing free or protected thiol groups; a solution containing 5 mol.% to 35 mol.% of a polymer containing vinyl groups; a buffer solution of alginate having a concentration of 1 wt.% to 4 wt.% suitable for receiving the biomaterial to be encapsulated; optionally, a calcium or barium salt for forming an ionic gelation bath; and optionally a reducing agent for an ionic bath or for post-printing gelation of a thiol-ene polymer. The kit can be used to produce a hydrogel column as defined herein, a three-dimensional hydrogel structure as defined herein, a graft as defined herein, or a hydrogel as defined herein.

[0013] Numerous other features and combinations thereof relating to the present improvement will be apparent to those skilled in the art after reading this disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is a graph showing the detailed fibrosis score (pericapsular hyperplasia-PCO) of two mice (Y1-1 and Y1-2) that received capsules formed of poly(methyl vinyl ether-alt-maleic anhydride) (PMM) with protected thiol (PMM-Spy) and side chain vinyl sulfone (PMM-VS) (combined in a ratio of 0.9:1.1). Capsules with 0-25% of their surface coated with PCO were classified into group (A), capsules with 25-50% PCO were classified into group (B), capsules with 50-75% PCO were classified into group (C), and capsules with 75-100% PCO were classified into group (D), as shown in Example 2.

[0015] Figure 2A is a graph showing in vitro insulin levels under the following conditions: A: free islets stimulated with 2.8 mM glucose, B: free islets stimulated with 28 mM, C: islets encapsulated in thiol-ene cross-linked cysteine-coated (TEC) polymer capsules stimulated with 2.8 mM, D: islets encapsulated in TEC polymer capsules stimulated with 28 mM, E: islets encapsulated in TEC polymer capsules stimulated with 2.8 mM (repeated twice), F: islets encapsulated in TEC capsules stimulated with 28 mM (repeated twice), as shown in Example 3.

[0016] Figure 2B is a graph showing blood glucose (BG) levels over time in STZ diabetic C57BL / 6j mice in which 900 islet equivalents (IEQ) of human donor islets were encapsulated in Figure 2A TEC capsules formed in, or blank capsules without islets, are marked as "islets" and "blank" in the figure, respectively, as shown in Example 3.

[0017] Figure 2C Yes, it shows acceptance Figure 2B Figure 3 shows the human C-peptide concentration in mice with encapsulated pancreatic islets "null" or "islets". They show that the level of human C-peptide in their circulation is higher than that of control mice (ie, null), as shown in Example 3.

[0018] Figure 3A is a fluorescent image of a calcium alginate (CA) TEC column having a length of 30 cm and prepared from 1.2% alginate and 1.5% 0.7:1.3 TEC as shown in Example 4.

[0019] Figure 3B is an image of a 1.2% alginate, 1.5% 0.7:1.3 TEC column loaded with approximately 61 IEQ / cm of rat islet clusters, as shown in Example 4.

[0020] Figure 3C is an image of a 1.2% alginate, 1.5% 0.7:1.3 TEC column loaded with approximately 61 IEQ / cm of rat islet clusters, as shown in Example 4.

[0021] Figure 3D It is a graph showing the average blood glucose concentration 11 days after transplantation of 15 cm of 1.2% alginate, 1.5% 0.7:1.3 TEC column fragments (loaded with approximately 61 IEQ / cm of rat islet clusters) (■) and 15 cm of blank 1.2% alginate, 1.5% 0.7:1.3 TEC column fragments (●) (n=4) in C57BL6 mice, as shown in Example 4.

[0022] Figure 3E Shown is an intact 15 cm segment of a blank 1.2% alginate, 1.5% 0.7:1.3 TEC column as described in Example 4 14 days after implantation in a C57BL6 mouse.

[0023] Figure 3F Shown are fragments of a broken 15 cm blank 1.2% alginate, 1.5% 0.7:1.3 TEC column 14 days after implantation in C57BL6 mice, as described in Example 4.

[0024] Figure 4 is a schematic diagram of post-bioprinting processing to obtain a 4-layer alginate patch, as shown in Example 4.

[0025] Figure 5A is a microscope image of a portion of a 1.0% CA-TE Cys30 polymer column (scale bar 500 μm), as shown in Example 4.

[0026] Figure 5B is a microscope image of a 1.0% CA-TE Cys30 polymer column encapsulating pancreatic islets (scale bar: 500 μm), as shown in Example 4.

[0027] Figure 5C is a microscope image of a 1.5% CA-TE polymer column before citrate treatment (scale bar 500 μm), as shown in Example 4.

[0028] Figure 5D is a microscope image of a 1.5% CA-TE polymer column after exposure to 70 mmol of citrate for 1 minute (scale bar 500 μm), as shown in Example 4.

[0029] Figure 5E is a microscope image of a 1.5% CA-TE polymer column after exposure to 70 mmol of citrate for 10 minutes (scale bar 500 μm), as shown in Example 4.

[0030] Fig. 5F is a microscope image of a 1.5% CA-TE polymer column after exposure to 70 mmol of citrate for 45 minutes (scale bar 500 μm), as shown in Example 4.

[0031] Figure 5G is a microscope image of a 1.0% CA-TE polymer column before citrate treatment (scale bar 500 μm), as shown in Example 4.

[0032] Figure 5H is a microscope image of a 1.0% CA-TE polymer column after exposure to 70 mmol of citrate for 1 minute (scale bar 500 μm), as shown in Example 4.

[0033] Fig.5I is a microscope image of a 1.0% CA-TE polymer column after exposure to 70 mmol of citrate for 10 minutes (scale bar 500 μm), as shown in Example 4.

[0034] Figure 5J is a microscope image of a 1.0% CA-TE polymer column after exposure to 70 mmol of citrate for 45 minutes (scale bar 500 μm), as shown in Example 4.

[0035] Figure 5K is a microscope image of a 0.75% CA-TE polymer column before citrate treatment (scale bar 500 μm), as shown in Example 4.

[0036] Figure 5Lis a microscope image of a 0.75% CA-TE polymer column after exposure to 70 mmol of citrate for 1 minute (scale bar 500 μm), as shown in Example 4.

[0037] Figure 5M is a microscope image of a 0.75% CA-TE polymer column after exposure to 70 mmol of citrate for 45 minutes (scale bar 500 μm), as shown in Example 4.

[0038] Figure 5N is a photograph of a 0.75% CA-TE polymer column before citrate treatment, as shown in Example 4.

[0039] Fig.5O is a photograph of a 1.0% CA-TE polymer column before citrate treatment, as shown in Example 4.

[0040] Figure 5P is a photograph of a 1.5% CA-TE polymer column before citrate treatment, as shown in Example 4.

[0041] Figure 5Q is a photograph of a 0.75% CA-TE polymer column after citrate treatment, as shown in Example 4.

[0042] Figure 5R is a photograph of a 1.0% CA-TE polymer column after citrate treatment, as shown in Example 4.

[0043] Figure 5S is a photograph of a 1.5% CA-TE polymer column after citrate treatment, as shown in Example 4.

[0044] Figure 5T is a live / dead fluorescent staining image of islets encapsulated in TEC columns before citrate treatment, as shown in Example 4.

[0045] Figure 5U is a live / dead fluorescence image of islets encapsulated in TEC columns after citrate treatment, as shown in Example 4.

[0046] Figure 5V This is an image of a 30 cm 1.0% CA-TE column (treated with 30 mM cysteine) before implantation in a healthy mouse, as shown in Example 4.

[0047] Figure 5W yes Figure 5W Image of the explanted column (25 cm section) as shown in Example 4.

[0048] Fig. 6Ais a photograph of a 1% calcium alginate CA-TE column treated with 30 mM cysteine ​​and encapsulating islets at 1 islet / inch (0.4 islets / cm), as shown in Example 4.

[0049] Figure 6B is a photograph of a 2% calcium alginate CA-TE column treated with 60 mM cysteine ​​and encapsulating islets at 2 islets / inch (0.8 islets / cm), as shown in Example 4.

[0050] Figure 6C is a microscope image of a 1% CA-TE column (treated with 30 mM cysteine) made from a solution containing 13,600 IEQ / mL (6.8 times), which yielded a column with 37 IEQ / cm, as shown in Example 4.

[0051] Fig.6D yes Figure 6C Photo of the pipe column.

[0052] Fig. 6E is a microscope image showing a 1% CA-TE (treated with 30 mM cysteine) column (n=10) with a diameter of 685±18 μm, as shown in Example 4.

[0053] Fig. 6F is a microscope image showing a 2% CA-TE (treated with 60 mM cysteine) column (n=10) with a diameter of 722±12 μm, as shown in Example 4.

[0054] Figure 6G Live / dead staining images of rat pancreatic islets after 4 days of encapsulation in 1% calcium alginate CA-TE columns treated with 30 mM cysteine, as shown in Example 4.

[0055] Figure 6H yes Figure 6G Close-up.

[0056] Fig. 7A is a phase contrast microscopy image of a 1% TEC Cys 30 mM column, as shown in Example 4.

[0057] Figure 7B is a bright field contrast microscope image of a 1% TEC Cys 30 mM column, as shown in Example 4.

[0058] Figure 7C is a bright field microscope image showing 1% TEC Cys 30 mM at the end of the column, as shown in Example 4.

[0059] Figure 8is a bar graph showing the mean insulin concentrations produced by human islets or islets encapsulated in TEC-cystamine columns on day 0 and day 4, as described in Example 7.

[0060] Fig. 9A is a microscopic image of pancreatic islets encapsulated in TEC-cystamine columns, as shown in Example 7.

[0061] Fig. 9B is a microscopic image of pancreatic islets encapsulated in TEC-cystamine capsules, as shown in Example 7.

[0062] Fig. 10A is a bar graph showing the average insulin concentrations produced by rat islets encapsulated in TEC-cystamine columns as described in Example 7 after 14 days post-transplantation (conditions labeled Ahr-1, Ahr-3) and 42 days post-transplantation (conditions labeled Ahr-4, Ahr-5 and Ahr-6).

[0063] Fig. 10B is a bar graph showing mean insulin release from rat islets encapsulated in TEC-cystamine columns as described in Example 7, 42 days after transplantation.

[0064] Fig.11A are microscopic images showing live / dead staining of pancreatic islets encapsulated in TEC-cystamine beads, as described in Example 7.

[0065] Fig. 11B are microscopic images comparing live / dead staining of encapsulated islets in TEC-cystamine beads and TEC-cystamine columns, as shown in Example 7.

[0066] Fig.12 is a graph showing the changes in blood glucose (BG) over time for transplanted blank TEC-cystamine columns and capsules, transplanted free islets, and transplanted islet-encapsulated TEC-cystamine columns and capsules, as shown in Example 7.

[0067] Fig.13 is a bar graph showing the mean insulin concentrations at time 0, 1, 2 and 3 hours for four different animals labeled (S35-5 to S35-8) transplanted with TEC-cystamine capsules encapsulating human islets after 29 days, as described in Example 7.

[0068] Fig.14A Live / dead staining of islets encapsulated in TEC-cystamine capsules 29 days after transplantation, as described in Example 7.

[0069] Fig. 14B is a live / dead staining of islets encapsulated secondarily in TEC-cystamine capsules 29 days after transplantation, as shown in Example 7.

[0070] Fig. 14C is a live / dead staining of the third encapsulated islet in TEC-cystamine capsule 29 days after transplantation, as shown in Example 7.

[0071] Fig.14D Yes Fig.14A Close-up of pancreatic islets.

[0072] Fig.14E Yes Fig. 14B Close-up of pancreatic islets.

[0073] Fig.14F Yes Fig. 14C Close-up of pancreatic islets.

[0074] Fig.15 is a photograph of a gel patch made from three layers of TEC-cystamine columns (no material was encapsulated), as shown in Example 7.

[0075] Fig.16A Live / dead staining of encapsulated islets in a gel patch made of TEC-cystamine columns with 2 wt.% alginate (scale bar 500 μm), as shown in Example 8.

[0076] Fig. 16B yes Fig.16A Wider view of the gel patch (scale bar, 2000 μm). DETAILED DESCRIPTION

[0077] Thiol-ene (TE) polymer hydrogels

[0078] Thiol-ene (TE) polymers are a pair of hydrophilic polymers, one functionalized with an activated olefin group and the other functionalized with a free or protected thiol such as SPy, which can form a cross-linked hydrogel when combined. Thiol / ene polymer pairs can form hydrogels alone, or they can provide enhanced robustness by forming a reinforcing network within a primary hydrogel gel such as calcium alginate or barium alginate. The network is formed by a thiol-ene addition reaction that can be triggered in the gelation or printing bath or in a separate subsequent step, such as by exposing the SPy-protected polymer thiol component to a reducing agent, such as the mild reducing agents tris(2-carboxyethyl)phosphine (TCEP) or tris(hydroxypropyl)phosphine (THPP), which releases free thiol groups on the thiol-functional vinyl polymer component (e.g., poly(methyl vinyl ether-alt-maleic anhydride) (PMM)). For example, once produced by protected thiol precursor (such as SPy), free side chain thiol groups will spontaneously couple with the vinyl sulfone groups on vinyl sulfone functional PMM in a few seconds to a few minutes, produce the PMM network of covalent crosslinking distributed in the whole calcium alginate hydrogel.In some embodiments, thiol-ene polymer network is defined as the polymer network of covalent crosslinking, wherein S atom covalently connects thiol component to one of carbon atoms (thiol-ene covalent bond) that was once a vinyl part.The formation of thiol-ene covalent bond is gelation or curing mechanism between thiol-containing polymer and vinyl-containing polymer.In some embodiments, one or both of thiol-containing polymer and vinyl-containing polymer are oxygen-containing polymers (for example, having at least two carbonyl groups in each repeating unit).Thiol group can be free or protected thiol group.This gelation or curing process and the thiol-ene polymer network formed are designed to be compatible with aqueous environment, and are compatible with the existence of living cells including mammalian cells and bacterial cells. These mammalian cells are usually selected for their therapeutic effects, and may include mammalian cells expressing hormones or enzymes, such as, but not limited to, insulin and coagulation factors, or bacterial cells, such as, but not limited to, natural or genetically modified Streptococcus thermophilus that are beneficial to intestinal diseases. These networks retain the high hydrophilicity of the thiol and ene polymer components that form the gel. Encapsulation of lactose-producing bacteria and other beneficial bacteria of the microbiome is contemplated herein. For example, a subject in need thereof can therefore receive oral administration of encapsulated bacteria, which are encapsulated in any of the systems described herein (including capsules, patches, and columns).

[0079] Thiol-ene is an advantageous gelation chemistry because it provides a covalently cross-linked gel, unlike chemistry such as pure ionic gelation or temperature-based gelation (e.g., gelatin and hydrolyzed collagen) that utilizes more easily broken physical cross-links. Thiol-ene cross-linking does not require potentially toxic catalysts used in gelation chemistry, such as copper-catalyzed azide / alkyne "click" chemistry. In addition, thiol-ene polymers can be prepared by fairly simple methods, and they retain their hydrophilicity, which is different from the polymers required for strain-promoted azide / alkyne click reactions. Covalently cured polymer networks are able to maintain their shape and integrity for a long time under in vivo conditions. This is in sharp contrast to some erodible ionically cross-linked networks (including calcium alginate alone). The permanent cross-linking nature of the network can prevent cells from being expelled from capsules, filaments or columns, and prevent immune cells from infiltrating the network. TE polymers are preferably combined with another hydrogel, such as alginate, elastin, hyaluronic acid, hydroxypropyl cellulose, carboxymethyl cellulose, methyl cellulose or gelatin. In some embodiments, the TE polymer accounts for more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95% or more than 99% of the weight of the polymer scaffold. In some embodiments, the polymer scaffold is essentially composed of alginate and TE or is composed of alginate and TE. In other embodiments, the polymer scaffold is essentially composed of TE or is composed of TE. In other embodiments, the polymer scaffold is formed by TE polymer and alginate, for example, with a weight ratio of 1:10 to 10:1, 1:5 to 5:1, 1:4 to 4:1, 1:3 to 3:1, or 1:2 to 2:1. In addition, in some embodiments, the hydrogel is further combined with a high molecular weight (MW) PMM polyampholyte (non-reactive) as a processing aid / filler. The MW of PMM can be changed by polymerization and / or oxidation to bridge between 80kDa and 1MDa.

[0080] Alginate hydrogels can be polymerized by ionic crosslinking, most commonly using calcium ions. However, other ions can be used instead of calcium, such as barium, strontium, copper, zinc, manganese, cobalt, lead, iron or aluminum. Alginate hydrogels can be formed at a concentration of 0.5wt.% to 8wt.%, 0.5wt.% to 6wt.%, 0.5wt.% to 4wt.%, 0.5wt.% to 3wt.% or 0.5wt.% to 2wt.%. For example, elastin or hyaluronic acid can be used in a concentration range similar to that of alginate.

[0081] In a preferred embodiment, the hydrogel is formed by thiol-ene polymer and alginate and optional hyaluronic acid. Hyaluronic acid is a non-fibrotic graft additive that can improve the strength and lubricity of hydrogel. When gel formation solution appears in the gel bath containing cation (such as calcium) and reducing agent (such as TCEP), the gelation of two kinds of network forming agents (alginate, thiol / ene) can occur simultaneously. Optionally, the gelation of thiol / ene network component can be carried out as a part of the subsequent exposure to independent TCEP gelling bath of the calcium alginate gel formed initially. Two networks reinforce each other so that the covalent network keeps physical integrity in long-term incubation and transplantation process. When there is, optional hyaluronic acid is physically trapped in the covalent network, and helps to provide hydrophilicity.

[0082] The hydrophilic backbone of the TE polymer can be a biocompatible polymer having a molecular weight of about 2000 to 2,000,000 Daltons, such as 5000 to 1,000,000 Daltons, or 20,000 to 500,000 Daltons. One polymer is a multifunctional Michael acceptor suitable for crosslinking with a second polymer that is a multifunctional Michael donor. Examples of suitable backbone polymers include, but are not limited to, homopolymers of polyacrylic acid or polymethacrylic acid, or copolymers of acrylic acid or methacrylic acid with anionic, uncharged or cationic monomers, including but not limited to styrene sulfonic acid; acrylamides and methacrylamides (such as acrylamide and N,N-dimethylacrylamide), or polyethylene glycol (PEG) acrylates and PEG methacrylates, wherein the degree of polymerization of the PEG side chains is 1 to 20 or more, and N,N-dimethylaminoethyl methacrylamide, N,N-dimethylaminoethyl methacrylate or acrylic anhydride. Other examples include polymers formed from copolymerization of alkyl vinyl ethers with anhydrides of dicarboxylic acids such as maleic acid, itaconic acid or citraconic acid, wherein the alkyl group consists of ethyl, n-propyl, isopropyl, n-butyl, sec-butyl or tert-butyl and higher alkyl groups (e.g., C5-C12); vinyl ethers of ethylene oxide oligomers with anhydrides (e.g., maleic anhydride or itaconic anhydride); polyanhydrides based on polyacrylic acid, which, upon dehydration, form cyclic anhydride moieties along the polymer backbone; polymers of carboxylic anhydrides such as acrylic anhydride, and copolymers of aromatic monomers such as styrene with maleic anhydride or other vinyl functional anhydrides such as itaconic anhydride or citraconic anhydride, for example alternating copolymers of styrene with vinyl functional anhydrides. Other backbone polymers include polymers containing epoxy groups such as glycidyl methacrylate and optionally neutral or anionic monomers selected from acrylamide and methacrylamide, N-alkyl substituted acrylamides and methacrylamides, hydroxyethyl acrylamide, hydroxyethyl methacrylamide, PEG methacrylate; and acrylic acid, methacrylic acid, vinylbenzenesulfonic acid and their respective alkali metal salts.

[0083] In some embodiments, the backbone polymer is a copolymer of an alkyl vinyl ether and an anhydride (such as maleic anhydride, itaconic anhydride or citraconic anhydride), such as poly(methyl vinyl ether-alt-maleic acid), because the anhydride group is easily functionalized and has biocompatibility by hydrolysis of the remaining anhydride portion after functionalization.

[0084] Olefin-polymers are backbone polymer side chains functionalized with crosslinkable compounds that react with the backbone polymer and contain activated olefin functional groups. The activated olefin functional groups may be, but are not limited to, vinyl sulfones, acrylates, methacrylates, maleimides, or alkynyls substituted with electron withdrawing groups such as esters.

[0085] Thiol-polymers are backbone polymer end chains or side chains functionalized with crosslinkable compounds containing free or protected thiol functional groups. The thiol functional group can be, but is not limited to, a thiol or a disulfide, such as a thiol protected by 2-pyridinethiol (SPy).

[0086] In one embodiment, the cross-linked hydrogel is formed by reacting an aqueous solution of a side chain functionalized main chain polymer of about 0.5wt.% to 15wt.% and preferably about 2.5wt.% to 7.5wt.% with an aqueous solution of a dithiol or polythiol cross-linking agent. Examples of suitable cross-linking agents include polar water-soluble compounds with two or more thiol groups, such as polyethylene glycol (PEG)-dithiol with a molecular weight in the range of about 200 to 1,000,000 Daltons, preferably a molecular weight of about 1000 to 20,000 Daltons. The cross-linking reaction involves the Michael addition of electron-rich nucleophiles (thiols) of side chain functionalized main chain polymers with a molar ratio ranging from 1:4 to 4:1 and preferably 1:2 to 2:1 with electron-poor olefins (e.g., vinyl sulfones, acrylates, maleimides). This addition reaction proceeds rapidly under physiological conditions, does not require a catalyst, and does not produce cytotoxic byproducts.

[0087] Theory predicts that an equimolar (1:1) ratio of thiols and alkenes should result in maximum cross-linking. It has been found that despite the use of an ideal 1:1 molar ratio of thiols to alkenes, a certain proportion of residual reactive groups (e.g., vinyl groups) remain on the network, physically limiting their participation in cross-linking. These residual groups carry the risk of subsequent reactions with unwanted biomolecules such as proteins during encapsulation and implantation, and thus immobilization of unwanted biomolecules, which may result in immune responses after implantation.

[0088] In particular, vinyl sulfones are strongly activated electrophiles that react with mobile thiol and amine functional groups on proteins present, for example, in the encapsulation solution or in the serum after transplantation. Such attached proteins are known to adopt unnatural conformations compared to circulating immune cell proteins. The side chain vinyl sulfone groups on the polymer backbone are inactivated by spontaneous hydration to form 2-hydroxyethyl sulfone groups, which are hydrophilic and largely benign groups, and the half-life of this addition of water to vinyl sulfone is about 2-3 days, which is too long to ensure complete conversion of residual vinyl sulfone at the time of transplantation (usually within 4 hours to 48 hours of encapsulation).

[0089] To alleviate the problem of residual groups, it was surprisingly found that the ratio of thiol: alkene can be adjusted to make alkenyls predominate to reduce or eliminate the formation of residual groups (especially free thiol groups). For example, the ratio of thiol: alkene is 0.95:1.05 to 0.5:1.5, preferably 0.95:1.05 to 0.65:1.35, or more preferably 0.9:1.1 to 0.7 to 1.3. Alternatively or additionally, a capping agent can be used to neutralize the residual groups to obtain modified residual groups. For example, post-functionalization can include deactivating residual alkenyls by adding deactivating moieties such as cysteine ​​or cysteamine or other thiols to deactivate reactive olefin sites.

[0090] In another embodiment, the cross-linked hydrogel is formed by reacting a side chain functionalized main-chain polymer (i.e., a first side chain functionalized main-chain polymer) comprising an activated olefin functional group with a second side chain functionalized main-chain polymer. The second side chain functionalized main-chain polymer is functionalized with a cross-linkable compound, which reacts with a cross-linkable group (e.g., vinyl sulfone) attached to the first main-chain polymer and comprises protected thiol. The main-chain polymer of the second side chain functionalized main-chain polymer can be as described above, and can be the same or different from the main-chain polymer of the first side chain functionalized main-chain polymer. Preferred main-chain polymers are copolymers of alkyl vinyl ethers and anhydrides such as maleic anhydride, itaconic anhydride or citraconic anhydride, such as poly (methyl vinyl ether-alternating-maleic anhydride).

[0091] The crosslinkable compound for preparing the second side chain functionalized main chain polymer will be combined with an entity that reacts with the main chain polymer in the first side chain functionalized main chain polymer, such as an amine group. The crosslinkable compound will also be combined with a protected thiol group. The protected thiol group is not particularly limited, and can be any group that can be easily deprotected to produce a thiol, which reacts with the reactive olefin of the first side chain functionalized main chain polymer to form a covalent bond between the first functionalized main chain polymer and the second functionalized main chain polymer. Examples of protected thiol groups that can be combined in the crosslinkable compound include but are not limited to disulfides, thiopyridines, dithiocarbonates, dithiocarbamates and thioesters. A preferred crosslinkable compound is S-(2-aminoethylthio)-2-thiopyridine. Another preferred crosslinkable compound is cystamine.

[0092] Cystamine incorporates a disulfide group, which represents a latent or protected thiol group that can be activated by the above-mentioned reducing agents and then used for crosslinking with other reactive polymers such as vinylsulfone-modified polymers. At the same time, compared to, for example, SPy groups, cystamine groups are considered to be more hydrophilic and therefore more cytocompatible and less likely to attract protein deposition in vivo (which could trigger further immune responses). Advantageously, reduction of the cystamine functional group generates a reactive thiol group without generating a hydrophobic small molecule byproduct.

[0093] Suitably, the thiol / ene network also provides the opportunity to attach a range of modified molecules by reacting with the original anhydride groups (in the case of amine functional molecules) and with residual thiol and / or ene groups. Such molecules may include attachment motifs (such as RGD), cationic groups (including primary or secondary amines, such as dimethylaminoalkylamines (C2-C5 alkyls, such as dimethylaminopropylamine or dimethylaminoethylamine), neutral groups with thiol moieties or other nucleophilic moieties (such as alcohols, such as aminoethanol); functional biomolecules (such as anti-inflammatory cytokines, cell-promoting proteins and growth factors), or small molecules (therapeutic agents, such as anti-inflammatory agents; detectable labels such as fluorescent labels, such as fluoresceinamine, TAMRA cadaverine, fluorescein cadaverine or rhodamine cadaverine, etc.).

[0094] Effect of thiol / ene composition on alginate demand

[0095] The addition of TE polymers provides an opportunity to reduce the concentration of sodium alginate in the polymer scaffold, because the viscosity of the alginate / thiol-ene polymer mixture increases compared to alginate alone, which helps beads or columns to form. In addition, the covalently cross-linked TE network formed in the CA gel means that the beads or columns will maintain their integrity. Therefore, in some embodiments, the present disclosure can effectively use a sodium alginate concentration of less than 1wt.% in the precursor solution. In the art, a concentration of 1wt.% of alginate is considered to be the lower limit of most applications of alginate hydrogels, especially in vivo. In the absence of any coating or thiol / ene enhancement, the calcium alginate gel traditionally formed by 1wt.% of sodium alginate is quite non-fibrous and may even disappear in vivo. Due to the thiol-ene cross-linked polymer of the enhanced alginate, when the alginate in the generated column or patch is lost by partial liquefaction and / or partial extraction after gelation, a stable hydrogel column or bead containing alginate far below 1wt.% can be formed. In one example, citrate can be used to extract alginate after extrusion and / or gelation. Thus, the concentration of alginate can be less than 1 wt.% alginate, less than 0.9 wt.%, less than 0.8 wt.%, less than 0.75 wt.%, less than 0.7 wt.%, less than 0.6 wt.%, or less than 0.5 wt.%. The advantage of reducing alginate to less than 1 wt.% is that it reduces the possibility of inducing a foreign body reaction through the immunogenic motifs inherent in the alginate itself, or through residual bacterial shell fragments, or other protein contamination that is not completely removed during the purification of this biological material from marine areas.

[0096] The hydrogels prepared so far do not rely solely on ionic crosslinking of sodium alginate with divalent ions such as calcium, strontium or barium to provide long-term mechanical integrity of the hydrogel matrix. Instead, the presence of sodium alginate helps:

[0097] a) act as a processing aid, in particular helping to protect cells from air shear and shear forces generated during extrusion, and

[0098] b) Maintaining the shape of the air sheared / extruded gel immediately after entering the gelling bath until covalent cross-linking occurs.

[0099] After covalent crosslinking, the ionic gelling component of the composite gel becomes less important. In the case of calcium loss after gelation, due to slow calcium / sodium exchange in tissue or intentional extraction of calcium (or strontium or barium, as the case may be) by reagents such as citrate or EDTA, part or all of the alginate lost by external diffusion will not be a concern for the stability of the hydrogel, and may even have an advantage because alginate has the potential for immunogenicity. Therefore, in one embodiment, alginate is extracted before transplantation. In order to be able to extract alginate, alginate of suitable molecular weight is used so that it can escape from the covalently crosslinked thiol / ene hydrogel network used. In addition, the gel formed using only 1wt.% of alginate (that is, without TE) is non-fibrotic in vivo, or dissolves in 2-4 weeks.

[0100] Columns and other three-dimensional implants

[0101] It has been found that when the gelling solution is continuously coextruded or extruded into a gelling bath, the thiol-ene and hydrogels described herein are advantageously formed into filament or tubular structures. In the context of the present invention, coextrusion can be understood to refer to providing two or more separate streams that are combined during extrusion, while extrusion can be understood as the extrusion of already combined components, i.e., a single stream containing a single composition. Since both techniques are applicable to the methods and processes of the present disclosure, when one term is used, the other term is also possible unless the context specifically indicates otherwise. As used herein, the term "filament" refers to the disclosed extruded hydrogel. The filament may be referred to as a "tubular column", and the term may be found herein and may be interchangeable. The filament may be a composite filament, and in one embodiment, a series of extrusion dies are placed close to each other so that the extruded tubular columns are attached to each other when they are extruded, so that the tubular columns form composite filaments in the shape of, for example, flat ribbons. In addition, the dies may have different cross-sections so that the filaments may be, for example, square tubular columns or oval tubular columns. In a preferred embodiment, the tubular column has a substantially circular cross-section.

[0102] In order to form long filaments or tubing, thiol-ene polymer is extruded into a gelling bath, which contains a reducing agent to induce the crosslinking of thiol-ene groups, and another reactant may also be included to induce the gelling of another hydrogel. For example, when the tubing is made of alginate and thiol-ene polymer, the gelling bath will contain a reducing agent to remove the protective group from thiol and allow thiol-ene crosslinking to occur, and contains cations (preferably calcium) to gel alginate. The solution to be gelled (precursor solution) is continuously extruded by a needle (preferably with a blunt end) and directly enters the gelling bath to produce the tubing. When referring to extrusion, as used herein, the term "directly" refers to the extrusion die (that is, in a preferred embodiment, the needle end) of the extruded content being immersed in a gelling solution or bath, or sufficiently close to the bath so that the extruded composition maintains its shape when it enters a gelling solution or bath. Suitably, the needle end can have a diameter less than 2000 μm, preferably less than 1000 μm, and more preferably less than 600 μm. The extruded hydrogel column can be considered to define a shell (i.e., outer diameter) and a core in which a biomaterial is encapsulated. The shell is denser than the core and encapsulates the contents in the core. Suitably, the core is an open network suitable for accommodating cells protected from the immune system by the polymer shell. The column can have an outer diameter of less than 2000 μm, preferably less than 1000 μm, and more preferably less than 600 μm. The size of the outer diameter is suitable for providing appropriate diffusion conditions for oxygen and chemicals to ensure that the cells in the encapsulated environment can survive with sufficient access to oxygen and can secrete and receive molecules passing through the polymer wall. In some embodiments, the column has a length of several centimeters, at least 10 cm, several meters or even longer. Therefore, the aspect ratio (length / diameter) of the column can be at least 5, at least 10, at least 20, at least 50, at least 100 or even larger. The dimensions (including length and diameter) of the column can be measured using a suitable microscopy technique (such as an optical microscope), and the aspect ratio is then calculated. The aspect ratio of these columns is defined as length divided by diameter. Other components, such as angiogenic agents and / or chemotactic agents, may be included in the shell, the core, or both. The method can be extended to include coaxial needles (e.g., concentric needles) to allow the formation of external and internal gel regions of the same or different compositions, such as an internal composition more suitable for cell support and an external region more suitable for providing mechanical strength and / or immune escape. Therefore, in some embodiments, the shell and core of the column have different compositions. For example, certain additives can be added in the shell, rather than in the core, and vice versa. In addition, the polymer composition of the shell and the core can vary.

[0103] The thiol / ene cross-linked network is formed by combining two mutually reactive polymers (thiol-containing polymer and vinyl-containing polymer) with alginate in the composite hydrogel. A general thiol-ene reaction (Scheme 1) is given below. Additional information on thiol-ene cross-linking is described in WO2018218346 (incorporated herein by reference in its entirety).

[0104] Solution 1.

[0105]

[0106] For example, both thiol / ene components are based on poly(methyl vinyl ether-alt-maleic anhydride) (PMM), modified in one case (PMM-Spy) with a protected thiol in the form of a Spy protected functional group (Scheme 2), and in the case of the other component (PMM-VS) with a side chain vinyl sulfone (Scheme 3). In another example, the protected thiol group is PMM-cystamine (Schemes 5-6).

[0107] PMM modification can be carried out in polar organic solvents (such as polar aprotic solvents), under conditions designed to introduce about 5mol.% to 35mol.% and preferably 10mol.% to 30mol.% and most preferably 15mol.% to 25mol.% of the corresponding thiol and vinyl into anhydride type PMM polymers. In some embodiments, the solvent is one of acetonitrile, N,N-dimethylformamide (DMF), tetrahydrofuran (THF), 1,4-dioxane, acetone and other such solvents known to those skilled in the art. The solvent should not contain nucleophiles, such as solvents that cannot be methanol or ethanol, because these two substances can react with the anhydride groups of the polymer starting material. As described above, thiol- and ene-functional PMM gel forming agents can be combined in different ratios, and can also be combined in different ratios of total TE polymers and sodium alginate (Scheme 4). The reaction occurs in the presence of a reducing agent to deprotect the pyridylthio group of PMM-SPy (or other PMM-thiols) to form a free thiol (SH) group that can react. An advantage of using PMM is that it is commercially available and inexpensive, and it is highly reactive toward amines and other nucleophiles, facilitating post-modification. Alternatives to PMM include, but are not limited to, copolymers of itaconic anhydride or citraconic anhydride with methyl vinyl ether, and copolymers of N-vinyl pyrrolidone with maleic anhydride or itaconic anhydride or citraconic anhydride. After functionalization, the remaining anhydride groups are hydrolyzed, rendering the resulting polyanionic gel former water-soluble.

[0108] Solution 2

[0109]

[0110] Solution 3

[0111]

[0112] Solution 4

[0113]

[0114] Solution 5

[0115]

[0116] Scheme 6. (Preparation of PMM-cystamine, see further discussion in Example 5 below)

[0117]

[0118] wherein R is H, a suitable optionally substituted alkyl group or another polymer linked via an amide bond.

[0119] As briefly explained above, the basic cross-linking chemistry of thiols (e.g., PMM-Spy) and vinyls (e.g., PMM-MS) involves a 1:1 ratio of deprotected thiols and vinyls. As previously known in the art, it is expected that this 1:1 ratio of functional groups will result in maximum cross-linking, and residual functional groups are minimal. Residual functional groups are considered to be undesirable because they may cause protein binding, thereby causing a foreign body reaction (FBR) of the hydrogel. Residual functional groups are also a problem of toxicity to encapsulated cells. In addition, the residual functional groups of thiols and vinyl sulfones are not hydrophilic / antifouling to the greatest extent, and therefore do not promote the hydration of the hydrogel to the greatest extent.

[0120] Surprisingly, it was found that deviating from a 1:1 ratio of thiol to alkene and favoring alkene groups can reduce residual groups. One advantage found is that adjusting the ratio of thiol to alkene, for example by adjusting the wt% of comparably functionalized PMM thiol and PMM vinyl, can optimize multiple parameters / aspects of the resulting hydrogel. Specifically, using an initial excess of alkene groups can allow for a higher conversion of a given amount of thiol groups, wherein the remaining excess alkene groups are subsequently capped by reaction with, for example, cysteamine, thereby generating hydrophilic and therefore beneficial betaine groups.

[0121] Variation of the functional group ratio of protected thiols and vinyl sulfones is an important tool to improve the final hydrogel properties with respect to optimizing the balance of mechanical robustness, hydration and swelling, antifouling properties, and permeability to nutrients and oxygen while blocking cellular and molecular immune components.

[0122] The ratio of thiol and alkene functional groups can be changed in two ways: by changing the functionalization degree of each in the two gel-forming PMM polymers, or by adjusting the weight ratio of the two polymers used, or a combination of the two methods. In practice, a simple method is to maintain the constant functionalization degree of both thiol (e.g., PMM-Spy or PMM-cystamine) and alkene (e.g., PMM-VS), and change the weight ratio of the polymer. The functionalization degree of each component is about 25 mol.%, which allows the two polymers to solubilize in aqueous media while making the cross-linked gel have enough mechanical robustness.

[0123] In one example, the SPy:CVS group ratio can be changed from 1:1 to 0.9:1.1, or even further to 0.7:1.3, or even 0.5:1.5, without a significant loss in final gel strength. The above examples retain the total loading of CVS+SPy polymer relative to the base formulation at a 1:1 ratio, although this is not necessary, for example, keeping SPy constant and increasing CVS is acceptable. It has been found that a total loading of 1.5 wt.% (0.75 wt.% of each) of CVS+SPy polymer is sufficient to form a permanently cross-linked hydrogel. The mechanical strength of the formed gel increases with increasing total loading of CVS+SPy. High loadings of gel forming agents (e.g., up to 3 wt.% of each) may be advantageous for encapsulating smaller cells, including, for example, therapeutic or genetically modified Streptococcus thermophilus or other bacteria for treating digestive tract disorders.

[0124] The gelation of one or more polymer networks forming the column can be controlled so that multiple columns extruded independently can be connected together by further gelation after extrusion. Any three-dimensional structure can be formed using this method. In one example, the gelling bath contains a reducing agent in an amount insufficient to induce complete gelation of the thiol-ene polymer. For example, the gelling bath may include a reducing agent (such as TCEP) of less than 80 mM, less than 50 mM, or 1 mM to 25 mM, but less than one equivalent relative to SPy, for example to provide a substoichiometric reaction of the SPy group. Even if there is only a limited level of calcium or barium column gelation, such as 5-50 mM or preferably 10-20 mM calcium, the partial ionic gelation of the column is affected, and covalent crosslinking is achieved by exposure to higher levels of calcium and barium, and exposure to TCEP or tris (hydroxypropyl) phosphine (THPP) to allow the overlapping column portions to be subsequently fused for a second time. Multiple extruded column structures can be positioned to form a desired three-dimensional geometry by only partially gelling the initially formed thiol-ene polymer column, and then further gelling by adding a sufficient amount of reducing agent, such as more than 1 equivalent of TCEP relative to SPy, or 1-5 equivalents, to allow deprotection of the remaining amount of SPy or other protecting groups. When combined by gelation, the resulting three-dimensional geometry is a single structure because the components are already covalently linked together. For example, extrusion through a set of closely spaced parallel nozzles can produce a ribbon or sheet formed from molten columns, where each individual column retains its ability to center cell clusters. These columns can be deposited in other shapes, including using templates, to impart the desired shape. One example would be placing the extruded column on a roller to impart a spiral shape. Another example would be depositing the columns into a multilayer porous sheet to further increase the packing density while still allowing for diffusion metabolic exchange through channels formed by empty spaces arranged in a two-dimensional cross-hatched pattern and potentially enhancing vascularization. In some embodiments, the hydrogel column is non-uniform (the shell is denser than the interior) to facilitate diffusion of oxygen and nutrients along the core of the column. In other words, the core has an open network structure, which allows oxygen, nutrients and other biomolecules to diffuse throughout the length of the column within the core. In some embodiments, this non-uniform column structure can be achieved by asymmetric gelation of the extruded column, or also by using coaxial extrusion. The column typically has a circular cross-section and is extruded by a circular needle, however, other shapes are contemplated herein, for example, a rectangular shape with a slit can be obtained, and square extrusion is also an option. In some embodiments, the column can be non-uniform along its length, for example, cells can be intermittently introduced into the extrusion stream (e.g., by using a T-joint in the extrusion system).In one embodiment, the goal is to have an outer shell that provides the molecular weight cutoff required to prevent the indiffusion of cytotoxic immune molecules (such as immunoglobulins), about 150,000 Daltons, but the density of the entire capsule is not high enough to interfere with the diffusion of nutrients and oxygen. This concept of only a small outer shell causing molecular weight restriction is standard for many technical membrane filters.

[0125] In the present disclosure, when referring to higher density, it should be noted that it is difficult to measure the actual solid content of density, so the present disclosure provides relative density instead (e.g., the shell is denser than the interior), which can be measured by the fluorescence intensity of a fluorescently labeled polymer (e.g., one polymer component is labeled, or alginate can be labeled). In one example, a hydrogel capsule or column or raft is prepared by fluorescently labeling one or more of a gel-forming polymer or alginate, and a spectral profile of fluorescence intensity on a hydrogel cross section is obtained using a confocal fluorescence microscope to measure the relative density of the hydrogel shell and the core region. Then, the fluorescence intensity is used as an indicator of the hydrogel density: a higher fluorescence intensity reflects a higher gel density, so the molecular weight cutoff is lower. The specific molecular weight cutoff can be measured in a separate experiment, where the hydrogel is immersed in a fluorescently labeled dextran solution of a specific molecular weight, and the internal diffusion rate and degree of these dextran molecules are determined by confocal fluorescence microscopy. It is known to those skilled in the art that there are conditions for preparing alginate hydrogels with a shell denser than the core. These include using a gelling bath or printing bath with an increased ratio of calcium chloride or barium chloride to sodium chloride.

[0126] In another example, during the extrusion process, the bath contains less than 1 equivalent of reducing agent or no reducing agent. The extruded non-crosslinked or partially crosslinked mixture can then be formed into a desired shape. Crosslinking is then initiated by exposure to a sufficient amount of reducing agent to gel the thiol and ene polymers. Optionally, a gelling aid such as gelatin and other thermogelling materials can be provided to facilitate gelation after extrusion.

[0127] The hydrogels and resulting structures described herein can be used to encapsulate biomaterials, such as cells, cell aggregates, cell spheroids or cell organoids. The term cell spheroid refers to cells aggregated into a spherical shape. This is different from organoids, because organoids mimic organ functions, and contain different cell types, and require vascularization. Mesenchymal stem cells (MSCs) (including primary MSCs, immortalized MSCs, differentiated cells and / or MSCs modified to overexpress appropriate mediators) are examples of cells that can be encapsulated by the methods described herein. Other examples include, but are not limited to, beta cells, islets, liver organoids, etc. In order to encapsulate cells, cell aggregates or organoids, these are included in a gelling solution before extrusion, and are extruded into the gelling solution by a blunt-end needle. This continuous extrusion process causes cell clusters (such as natural islets and islet reaggregates and stem cell clusters) to flow-induce centering in the center of the extruded tubing column. This keeps the encapsulated cell clusters / islets away from the wall of the gel tubing column, thereby enhancing their physical immune protection. This feature is attributed to the flow dynamics applied by the needle size, solution viscosity and cell cluster size. Other methods to ensure that cells and / or biological agents are centered can include coaxially extruding the active ingredients in the flow of the core with an inert gel-forming agent extruded by an annular flow. The end of the column is made of a denser shell to completely encapsulate the core and protect any cells encapsulated in the shell from attack by the immune system. In some embodiments, the end of the column can be capped to improve immune isolation. The column can be transplanted to the subject alone, or transplanted with a support, or can be transplanted as part of the device. The peritoneal cavity and preferably the omentum bag are suitable locations for the transplant. In one embodiment, the column is placed in the omentum bag and sutured to form an integral part of the omentum bag. Therefore, these columns are usually physically combined or encapsulated in the omentum bag. In some embodiments, nylon surgical mesh or other suitable graft supports can be used to suture the column to the tissue, such as the omentum bag. Alternatively, tissue glue (e.g., fibrinogen or thrombin glue) can be used to stabilize the column on the tissue. The use of supports can be problematic as it can increase fibrosis and reduce oxygen diffusion into and out of the string, therefore, in some embodiments, the string or 3D structure formed therefrom is used without a support substrate. Surprisingly, it was found that the hydrogel string is compatible with syringe injection. A suspension containing one or more hydrogel strings can be stored in a syringe and the strings are observed to leave the nozzle without tangling. In one embodiment, the syringe absorbs the string from a first end and then dispenses the string from a second end (the last end to enter the syringe). In a preferred embodiment, a portion of the string in the syringe is located in the needle portion of the syringe and the remainder of the string is loosely coiled in a container of the syringe with a flushing solution (e.g., saline).

[0128] Benefits of the polymeric columns described herein may include, but are not limited to, ease of retrieval, particularly for smaller cell numbers, having encapsulated islets or other cell clusters and spheroids located in the center of the column, the ability to be pre- or post-modified with desired attachment functionalities or other biomolecules, and long-term integrity due to the cross-linked TE network that allows for multiple media exchanges without weakening the hydrogel.

[0129] Column extrusion can be performed in a full strength gelling bath containing 100 mM calcium chloride, which causes a calcium alginate skin to form instantaneously on the nascent column, preventing the column segments from adhering to each other. This mimics the process of preventing beads from adhering in the gelling bath and helps form long smooth column sections.

[0130] In contrast, the calcium chloride concentration in the gelling bath is reduced to about 25mM calcium chloride, which allows the tubular column fragments formed within a few seconds of each other to adhere to each other, thereby achieving partial fusion of the overlapping parts of the tubular column. This helps, for example, 3D bioprinting of cross-hatched patches of tubular columns (a pattern that can be used to compactly assemble tubular columns containing cell clusters). Such patches are still able to perform metabolic exchange through passive diffusion and vascularization of channels within the patch. In order to achieve such an embodiment, the calcium chloride level in the gelling bath can be reduced from the standard 100mM to 10mM to 50mM. Such cross-hatched patches can be extended to three dimensions, for example, by overprinting two or more cross-hatched patterns on top of each other, provided that the open spaces are fully arranged to become channels for metabolic exchange through passive diffusion and final vascularization. Similarly, the TCEP level in the gelling bath can be reduced to a point where gelation is delayed enough to achieve partial fusion of overlapping tubular columns. An example is to reduce the TCEP level to zero during the formation of the tubular column or patch, and to initiate thiol / ene crosslinking by exposing the sheet to TCEP afterward in a separate step. This may include incorporation of other gelling aids such as gelatin and other thermogelling materials.

[0131] In some embodiments, the hydrogel column is inhomogeneous, for example, having a higher network density on the surface of the column. This can be achieved by coating or enhancing the outer surface of the hydrogel column with polycations after gelation, further crosslinking, and introducing hydrophobicity. Other methods may include inhomogeneous gelation, such as by using a sodium-free calcium gelling bath, or indeed by using coaxial extrusion. This may increase surface strength and facilitate lateral (along the center of the column) diffusion of oxygen and nutrients and possible cells. In one embodiment, this will allow the encapsulation of single cells or small cell clusters, which then expand and self-aggregate into a "cell column" along the center of the hydrogel column, thereby possibly optimizing cell loading and metabolic connectivity. When the column is inhomogeneous (the shell is denser than the column core), this maximizes the relationship between strength and gel loading.

[0132] Example

[0133] Example 1: Thiol-ene cross-linked network synthesis

[0134] The thiol / ene cross-linked network was formed by combining two mutually reactive polymers with alginate in the composite hydrogel. Both thiol / ene components were based on 80 kDa poly(methyl vinyl ether-alt-maleic anhydride) (PMM), modified with Spy-protected thiols in one case (PMM-SPy) and with pendant vinyl sulfones in the case of the other component (PMM-VS).

[0135] These PMM modifications were carried out in acetonitrile solution under conditions designed to introduce about 5 mol.% to 35 mol.% and preferably 10 mol.% to 30 mol.% and most preferably 15 mol.% to 25 mol.% of the corresponding Spy and ene (Ene) groups into the anhydride form of the PMM polymer. Briefly, the anhydride form of the PMM (1 g, 6.41 mmol of anhydride) was dissolved in 20 mL of acetonitrile and transferred to a round-bottom flask equipped with a magnetic stirring bar. Triethylamine (TEA, 360 uL) was added dropwise to the vigorously stirred reaction mixture, followed by S-(2)-pyridylthiocysteamine hydrochloride (0.3 g; 1.35 mmol) or cysteamine vinyl sulfone hydrochloride (0.36 g; 1.55 mmol) to prepare PMM-SPy or PMM-VS, respectively. After overnight reaction, the reaction mixture was transferred to 6-8 kDa molecular weight cutoff tubing and dialyzed in a water bath containing 1 wt.% sodium chloride, with a total of 2 changes per day for 2 days, followed by 2 changes per day for 2 days. The dialyzate was subjected to UV-visible absorbance measurement to confirm that no small molecule impurities were detected. The dialyzed polymer solution was freeze-dried to obtain PMM-SPy and PMM-VS in the form of light pink solids. 1 H NMR was used to confirm that the modification percentage was about 20 mol%.

[0136] To prepare TEC 0.9:1.1 (TEC 911) capsules, PMM-Spy and PMM-VS were combined in a weight ratio of 0.9:1.1, respectively, while maintaining a total polymer loading (i.e., 2% total polymer loading). Before proceeding to form the column, capsules were first prepared to test chemical properties. PMM-SPy (36.0 mg) and PMM-VS (44.0 mg) were dissolved in 1.80 mL of 35 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) buffered saline (HBS) and adjusted to pH 7.6 with 1 M sodium hydroxide. After pH adjustment, the total volume of the polymer solution was increased to 2.00 mL by adding 35 mM HEPES buffered saline. The polymer solution was mixed with 2.00 mL of 2 wt.% sodium alginate (Novamatrix PRONOVA TM The mixture was filtered through a sterile filter of 0.22 μm and then transferred to a 3 mL syringe, which was equipped with a 20 G, 14 G outer coaxial needle (Ramé-Hart Instrument Co.), and placed in a vertical guide syringe pump (Harvard Apparatus Pump 11 Elite) in a biosafety cabinet. Polymer solution was extruded into the aseptic gelling bath solution of 50 mL of continuous stirring by an inner needle to prepare capsules, which contained 100 mM calcium chloride, 35 mM HEPES buffer, 0.45 wt.% sodium chloride and 3.5 mM tris (carboxyethyl) phosphine hydrochloride (TCEP) adjusted to pH 7.6. The liquid extrusion rate by the inner needle was set to 15 mL / H, and the air flow rate by the outer needle was set to 2.5 L / min, and the tip of the inner needle was wiped once in every 1.5 minutes to remove any dry alginate. The capsules were treated by continuous vortexing for 10 minutes in a 30 mM L-cysteine ​​and 3.5 mM TCEP solution adjusted to pH 7.6, followed by two washes with saline. After extrusion, the capsules were placed in a gelling bath for a total of 15 min, followed by two washes with saline. The capsules were suspended at a 1:1 v / v capsule: saline ratio for implantation.

[0137] Extra PMM-VS allows the conversion rate of the thiol group released from PMM-Spy to increase. This also leads to the presence of excessive residual PMM-VS functional groups, which can then advantageously react with post-modification agents such as cysteine ​​to introduce a corresponding larger proportion of antifouling functional groups. Other relevant ratios determined are 0.8:1.2 and 0.7:1.3 and 0.5:1.5. These maintain a constant overall polymer loading, and therefore maintain gel formation agent viscosity (from the perspective of microfluid flow characteristics, cell shear stress and 3D printing, this is required).

[0138] Example 2: Transplantation of TEC 0.9:1.1 into healthy mice

[0139] This example provides evidence of the antifouling functional groups of the implant according to an embodiment of the present invention. PMM-Spy and PMM-VS were combined in a ratio of 0.9:1.1 to form blank capsules, which were treated with 30mM cysteine ​​and transplanted into the intraperitoneal space (IP) of healthy C57BL / 6 mice. After two weeks, the capsules were explanted with a recovery rate of 50-80% and found to have minimal fibrosis. Figure 1 Detailed fibrosis results are summarized.

[0140] Example 3: Islet encapsulation and cell function of 0.9:1.1 TEC and in vivo immune protection

[0141] This example provides evidence of in vitro and in vivo cell function when encapsulated in a 0.9:1.1 thiol:ene hydrogel. The formulation was found to improve insulin release by glucose-stimulated insulin secretion (GSIS) compared to a 1:1 thiol:ene hydrogel or alginate hydrogel alone. Glucose stimulation triggered a two-fold improvement in insulin release, suggesting that rapid mediator exchange ( Figure 2A ), and in vivo, it maintained human islet function for up to 50 days (which is longer than that reported for free non-encapsulated islets in the literature (e.g., Qi, M., Y.,Lacík,I.,Formo,K.,Marchese,E.,Wang,Y.,...&Strand,BL(2012).Survival of human islets in microbeadscontaining high gluronic acid alginate crosslinked with Ca 2+ and Ba 2+ .Xenotransplantation,19(6),355-364, and Qi,M.,Strand,BL, Y.,Lacík,I.,Wang,Y.,Salehi,P.,...&Oberholzer,J.(2008).Encapsulation of human islets innovel inhomogeneous alginate-ca 2+ / ba 2+microbeads: in vitro and in vivo function. Artificial cells, blood substitutes, and biotechnology, 36(5), 403-420), i.e., extended by up to 8 times), as shown, hyperglycemia in diabetic immunocompetent mice rapidly recovered to normoglycemia and lasted for about 50 days ( Figure 2B ), and elevated human C-peptide levels in the serum of immunocompetent diabetic mice receiving human islets encapsulated in a 0.9:1.1 thiol:ene hydrogel formulation treated with cysteine ​​( Figure 2C The cells remained functional and reduced blood glucose to normal levels (200 mg / dL + / - 50 mg / dL SD) for up to 68 days ( Figure 2B ).

[0142] Example 4: TEC column

[0143] The formulations (thiol:ene ratios of 0.9:1.1, 0.8:1.2, and 0.7:1.3) were extruded into a gelling bath in a continuous manner to form columns. These columns are an advantage for retrievable implantable devices, as cell clusters are centered by microfluidic effects, which can better prevent cell-cell contact, and have other processing advantages, such as avoiding the need for constant sterile airflow. The embodiments include gel former solutions containing 0.5-4wt.% sodium alginate and 0.5-6wt.% of each of the thiol and ene polymers. The formation of continuous gel columns greater than 1 m in length and 0.2 mm to 1.5 mm in diameter was demonstrated. Cysteine ​​treatment (first washing in saline, then exposing the column to 10-60 mM cysteine ​​in HBS) was performed to convert any remaining vinyl sulfone groups to non-reactive groups, thereby obtaining thiol-ene cysteine ​​(TEC) polymers. Figure 3A Images of cysteine ​​treated fluorescent 0.55 mm diameter 1.2% alginate, 1.5% 0.7:1.3TEC columns greater than 1 m in length are shown. It is further shown that these columns can bind rat islets that have survived in vitro for several days. Notably, these rat islets appear to be consistently located in the center of the extruded column ( Figure 3B-3C TEC columns containing rat islets were transplanted into the peritoneal cavity of diabetic mice by injection, and a short-term reduction in blood glucose levels (up to 11 days) was observed compared to mice receiving blank TEC columns ( Figure 3D ). Finally, it has been shown that after transplantation of TEC columns into the peritoneal cavity of immunocompetent mice and quantitative explantation two weeks later, the explanted columns showed little to no cell attachment along the length of the column. Some column fractures were observed, with more pronounced fibrosis accumulating at the fracture ends, which was due to the roughness of the fracture edges ( Figure 3E-3F ). The string is easily transplanted into the intraperitoneal injection (IP) space. This breakage can be mitigated by incorporating additives such as hyaluronic acid to improve lubricity without reducing strength. Furthermore, in large animals and indeed humans, transplantation into an omental bag will involve abdominal insufflation, which will reduce the risk of string damage during injection.

[0144] Although the tubing is a prototype of a retrievable device, the tubing length required to transplant a therapeutic dose of islets is about 15 cm for mice (1000 IEQ / 25 g mice) and may be up to 40 m or longer for humans (about 300,000 IEQ / 75 kg humans). Therefore, tubing extrusion is adjusted to form a patch that combines a high surface area (required for islet survival and good metabolic connectivity) and a compressed form factor. This approach produces a palm-sized patch containing a therapeutic dose of islets for humans. The microporosity of the patch is adjusted to maximize vascularization and integration with host tissue. The advantages of the present device include TE and TEC compositions, as well as the centering of islets in the tubing.

[0145] An advantageous feature of the column formation is the ability to microfluidically center the islets and ensure strong fusion between chains of overlapping columns extruded after 3D bioprinting into a 3D patch device. This is difficult to achieve because conventional chemistry is designed to gel by calcium immediately when the columns enter the gelling bath, so that the overlap of new columns with columns extruded even seconds ago will be compromised by the formation of a gel skin on the outer surface of the earlier column sections. The inventors have developed a method to overcome this. One method is to print the scaffold directly into a patch-shaped female mold containing a gelling bath solution, which will inhibit the columns from moving around to undesirable positions during the printing process. Another method is to print the columns into a viscous support bath (e.g., FRESH TM(freeform reversible embedding of suspended hydrogel) material or equivalent), this support bath can support and maintain the shape of the printed material until a solution can be added to induce crosslinking of the alginate or TEC polymers (using CaCl2 or a reducing agent, TCEP, respectively). Another approach is to print a supporting biomaterial in the TEC / alginate formulation along with or even in place of alginate. Examples of such materials include gelatin, Pluronic F127, etc., which can be bioprinted on a variety of surfaces without the use of a supporting bath, where the material itself supports the shape of the printed material until crosslinking of the alginate or TEC is initiated by the addition of CaCl2 or TCEP, respectively. Importantly, if desired, these materials can completely replace alginate in the formulation and can be removed after crosslinking. For example, specific concentrations of gelatin or Pluronic F127 can be removed by heating the patch to 37°C or cooling it to 4°C, respectively. Proof of concept for this last strategy is shown in Figure 4 shown. Figure 4 A bioprinted 4-layer cross-hatched patch composed of alginate and gelatin is shown, which was printed on a petri dish using an inexpensive modified commercial 3D printer. The gelatin here supports the 3D structure in a dry state throughout the printing process, and after printing is completed, it is soaked in a CaCl2 solution for 15min to cross-link the alginate, and then the patch is removed and placed in a solution of distilled water or saline at high temperature (37°C) to remove most of the gelatin, leaving behind an alginate patch. Similar methods can be used to produce implantable patches using gelation, Pluronic F127, etc., using TEC alone (its cross-linking mechanism also works within seconds), TEC / alginate, or TEC with other additives. Similar methods can be used to obtain similar 100% TEC patch formats. In addition, the extruded overlapping column sections can be held in place by the presence of reduced concentrations of calcium in the main extrusion bath, such as 1-50 or preferably 2-20mM, which is sufficient to retain the extruded column segments, but still low enough to enable the overlapping column segments to fuse. Once patch extrusion is complete, exposure to THPC triggers covalent crosslinking to permanently lock the string segments in place. Finally, a substoichiometric amount of THPC reducing agent can be added to the main gelling bath to initiate partial gelation upon extrusion but retain the majority of the crosslinking functionality for post-gelation after extrusion is complete.

[0146] The column formation was performed by extrusion with a syringe pump, preferably with a blunt tip. Islets were encapsulated using TEC columns with 1% or 2% CA-TE (with 30 mM or 60 mM cysteine, labeled CA-Tex / CysY, where x = percentage of CA-TE and Y = mM of cysteine) at 1 or 2 islets / inch. Figure 5A-5B The sharp edge of the "cut" column end is shown, indicating that radial inward diffusion of calcium leads to radial density gradients in both the calcium alginate and TE network (using CA-TE1.0 / Cys30). Unless otherwise stated, when a percentage is specified in relation to CA-TE, it refers to CA fixed at 1% TE concentration. A combination of good strength and good permeability throughout the column shell and inside the column shell was obtained. Swelling of the columns was observed for 0.75%, 1.0% and 1.5% TE after up to 45 min of citrate challenge ( Figure 5C-5M As shown in Table 1, 340-450% swelling was observed. The mechanical robustness of the columns with 0.75%, 1.0%, and 1.5% TE before and after citrate exposure was compared ( Figure 5N-5S ). Before citrate exposure, at 0.75% TE, the column broke at 4 cm when lifted with tweezers, and at 1.0% and 1.5% TE, the entire length was supported by the tweezers. After citrate exposure, the column broke at 1 cm, 3 cm, and 5 cm for 0.75%, 1%, and 1.5% TE, respectively. After stripping calcium from alginate, the TEC column was confirmed to be mechanically stable and maintained structure ( Figure 5T-5U ). Examples of islet-encapsulated tubular columns include Figure 5V-5W As shown. Place 30 cm of CA-TE 1％ (Cell-free CYS 30mM Tube) were injected intraperitoneally into healthy mice and retrieved 7 days later.

[0147] Table 1 .TE swelling measurement

[0148]

[0149] The appearance of the column is as follows Figures 6A-6E For CA-TE1.0 / Cys30, two islets per inch were obtained, and for CA-TE2.0 / Cys60, one islet per inch was obtained ( Figures 6A-6D For CA-TE1.0 / Cys30, islets were centered and survived in vitro for up to 1 week within a diameter range of approximately 700 μm ( Figure 6E-6F The viability of cells was indicated by green fluorescence of live / dead staining ( Figure 6G-6H). Rat islets were stained with phenanthridin / calcein AM live / dead staining 4 days after encapsulation. In addition, these were transplanted intraperitoneally in STZ diabetic mice. Blood glucose control was observed for a week (Table 2). Within 5 days, blood glucose dropped, and although this was a temporary effect, it lasted for a week. The strength of the tubing in the intraperitoneal space of healthy mice was also tested. After 2 weeks, the tubing explanted from healthy mice transplanted intraperitoneally 2 weeks ago was retrieved well, but appeared shorter and broken at the end. This is attributed to the small intraperitoneal injection space injected into mice without the benefit of inflation, which would alleviate this problem in large animals and indeed humans.

[0150] Table 2 Blood glucose levels in mice that received the tubular implants

[0151]

[0152] Columns with a diameter of 566 μm (SD 6 μm) made from a 1% TEC Cys 30 mM composition were used to encapsulate stem cell clusters with a diameter of 54 μm (SD 15 μm). Table 3 summarizes the characteristics of the columns used. When the column was extruded, it was noted that the cell clusters were well centered ( Figures 7A-7C ).

[0153] Table 3 .Characteristics of the pipe string

[0154]

[0155] SD = Standard Deviation

[0156] Example 5: Synthesis of PMM-cystamine

[0157] In another example, PMM-cystamine (PMM-Cy) is used as a precursor of reactive thiol polymers, rather than PMM-SPy. In order to prepare PMM-cystamine targeting 10mol% functionality, PMM (1g, 6.41mmol anhydride) in anhydride form is dissolved in 75mL of N,N-dimethylformamide (DMF) and transferred to a round-bottom flask equipped with a magnetic stirring bar. Triethylamine (TEA, 360uL) is added to the vigorously stirred reaction mixture, followed by dropwise addition of cystamine dihydrochloride (0.144g, 0.64mmol) in 4mL of 1:1v / v DMSO: A solution in DMF. After overnight reaction, the reaction mixture is transferred to 12-14kDa molecular weight cut-off tubing and placed in a water bath containing 2wt.% sodium chloride distilled water for dialysis, totaling 2 changes per day for 2 days, followed by 2 changes per day of distilled water baths for 1 day. The dialyzate was subjected to UV-visible absorbance measurement to confirm that no small molecule impurities were detected. The dialyzed polymer solution was freeze-dried to obtain PMM-cystamine in the form of a light pink solid. 1 H nuclear magnetic resonance (NMR) was used to confirm the purity of the polymer, and no small molecule impurities were detected. The sample was subjected to elemental analysis, and 1.5% N, 38.28% C, 5.03% H and 3.63% S were obtained, and the cystamine unit of the PMM main chain was calculated to be about 13% modified. It should be noted that the reaction conditions, especially the reaction concentration, play a key role in the formation of PMM-cystamine as a soluble product. Under concentrated conditions (i.e., a concentration of more than 150mM PMMAn), cystamine reacts with PMMAn to form a higher probability of chain amide bonds between polymers, which effectively forms crosslinks (the second row of compounds on the right side of Scheme 6) to produce insoluble products. By diluting the reaction mixture (i.e., diluting with a 2.5- to 4-fold dilution factor of the aforementioned concentrated concentration), cystamine is more likely to react with the chain anhydride groups in the polymer, thereby producing macrocyclic amide bonds, which produces soluble products (the second row of compounds in the middle of Scheme 6). The structure shown in the middle compound in the second row of Scheme 6 is an example of reaction with adjacent anhydride groups, however larger macrocycles can also react with anhydride groups on the same polymer chain that are further away. It is possible that cystamine is monosubstituted and reacts with a single anhydride to produce a side chain amine (second row of compounds on the left side of Scheme 6), however this outcome is unlikely because the reaction was left overnight and there were excess anhydride groups available for reaction. Reactions that produce macrocycles are preferred because this produces a soluble product and there are also two reactive polymer thiols per mole of cystamine after reduction, which can be used to form thiol-ene bonds in the subsequent gelation reaction. The reaction conditions described were found to be optimal for the formation of soluble PMM-cystamine (as a gel former).

[0158] Example 6: Preparation of TEC-cy gel-forming solution to prepare hydrogel capsules, columns and patches

[0159] To prepare TEC-cy hydrogel capsules, PMM-cystamine (10mol% functionality) and PMM-VS (20mol% functionality) are combined in a 1:1 molar ratio of thiol to vinyl, while targeting a total wt. / v% of preferably 1.5%, and a final concentration of 1% sodium alginate. For example, to prepare 4mL of TEC-cy gel-forming solution, PMM-cystamine (28.3mg) and PMM-VS (31.7mg) are dissolved in 2mL of HBS, and the pH is adjusted to pH=7.4 with 1M NaOH. 2mL of 2% sodium alginate is added to the solution and vortexed to form a final concentration of 1.5% thiol-ene polymer and 1% sodium alginate. Before forming hydrogel capsules, columns, and patches, the gel-forming mixture is filtered using a 0.22μm syringe filter.

[0160] Formation of TEC-cy hydrogel capsules

[0161] In order to prepare TEC-cy hydrogel capsules, the TEC-cy gel formation solution described above is loaded into a syringe, and is equipped with a 20G, 14G outer coaxial needle (Ramé-Hart Instrument Co.), and is placed in a vertical guide syringe pump (Harvard Apparatus Pump11Elite) in a biosafety cabinet. Capsules are prepared by extruding the polymer solution through an inner needle into a gelling bath solution containing 100mM calcium chloride, 35mM HEPES buffer, and 0.45wt.% sodium chloride. The liquid extrusion rate through the inner needle is set to 15mL / h, and the air flow through the outer needle is set to 2.7L / min, and the tip of the inner needle is wiped once every 1.5min to remove any dry alginate. After extrusion is completed, the capsule is cured in a gelling bath for 15min. The capsule is transferred to a TCEP solution to cleave the disulfide group and initiate covalent crosslinking by the reaction of thiol-ene and vinyl sulfone groups. The capsules were treated by continuous vortexing for 10 min in a 25 mM L-cysteine ​​solution adjusted to pH 7.6, followed by two washes with saline. After extrusion, the capsules were placed in a gelling bath for a total of 15 min, followed by two washes with saline. The capsules were suspended at a 1:1 v / v capsule: saline ratio for implantation.

[0162] Formation of TEC-cy hydrogel columns

[0163] To prepare the TEC-cy hydrogel column, the TEC-cy gel-forming solution was loaded into a syringe and equipped with a 20G blunt needle. The syringe was mounted in a vertically oriented syringe pump and the needle tip was immersed in a gelling bath containing 100 mM calcium chloride, 35 mM HEPES and 0.45 wt.% sodium chloride solution at pH 7.4, or a barium gelling bath containing 10 mM barium chloride, 35 mM HEPES and 0.45 wt.% sodium chloride at pH 7.4. The polymer solution was extruded into the gelling bath at a liquid flow rate of 10 mL / h.

[0164] Example 7: Encapsulation of Pancreatic Islets

[0165] Encapsulation of pancreatic islets in TEC-cy capsules

[0166] In order to prepare capsules containing islets, first 2wt.% sodium alginate in HBS was diluted to a concentration of 1.5wt.% by adding HBS, and then mixed with a pipette for 5min. The resulting alginate solution was aseptically filtered (0.22 μm filter), and then further diluted with the islet suspension in HBS to form a final concentration of 1wt.% sodium alginate (containing islets). The alginate-islet mixture was gently mixed with a pipette for 5min. The suspension was transferred to a 1mL syringe and then loaded into a syringe pump with a liquid flow rate of 15mL / hour. The syringe was equipped with a 20G / 14G coaxial flow blunt needle (Ramé-Hart), and the side arm was connected to the air line, and the air flow rate was adjusted to 2.5L / min. The sodium alginate solution containing islets was extruded into a gelling bath solution stirred at pH 7.4, which contained 100mM calcium chloride, 77mM sodium chloride, and 35mM HEPES. After extrusion, the beads were collected into 50 mL centrifuge tubes and allowed to solidify on ice for 15 min with occasional mixing. After incubation, the beads were allowed to settle to remove the supernatant. Prior to the coating procedure, the beads were washed twice with saline at a 3:1 bead to solution ratio as described in the following section.

[0167] Transplantation into C57Bl / 6J mice

[0168] C57Bl / 6J mice were weighed and anesthetized with isoflurane. After complete anesthesia, animals were shaved along the abdomen and cleaned with alcohol wet paper to prepare for intraperitoneal injection. Under anesthesia, animals were subcutaneously injected with buprenorphine (0.05mg / kg) for analgesia, and intradermal injection of bupivacaine (8mg / kg) was used for local anesthesia at the injection site. Then, animals received the dose (total injection volume was 1mL) of encapsulated islets (800IEQ) or blank capsules in 0.9% sodium chloride (saline) injected intraperitoneally with a 16G needle. The syringe was loaded with 1-2mL of saline after injection, and ejected into a clean 50mL tube to collect any remaining capsules. Animals were recovered on a heating pad, and observed for any discomfort.

[0169] At the end of the experiment, the animals were humanely euthanized and blood was collected by cardiac puncture. The capsules were explanted, the peritoneum was washed with 0.9% NaCl (saline), and placed in a stainless steel kidney collection dish. Using a plastic Pasteur pipette, the capsules were collected into a fresh 50 mL test tube. The capsules were washed twice with fresh 0.9% NaCl (saline) to remove any debris or unattached cells. The capsules were then transferred to 4% v / v formalin (methanol-free) to a ratio of 10:1 formalin solution to capsule. The capsules were then imaged for pericapsular fibrosis (PCO) analysis as shown below.

[0170] To measure islet function by insulin secretion, low (2.8 mM) and high (16.7 mM) D-glucose Krebs-Ringer bicarbonate HEPES (KRBH) buffer (135 mM NaCl, 3.6 mM KCl, 5 mM NaHCO3, 0.5 mM NaH2PO4.2H2O, 0.5 mM MgCl2.6H2O, 1.5 mM CaCl2.2H2O, 10 mM HEPES, and 0.1% BSA) at pH 7.4 were prepared and sterile filtered using a 0.22 μm filter before use.

[0171] For each round of islets and encapsulation, islets were stimulated in vitro in 24-well sterile plates. Briefly, 10 free islets or 10 capsules with islets having a diameter of about 100 μm were distributed in 24-well plates (10 islets / well) in triplicate in 1 mL of warm supplemented RPMI 1640 culture medium. Free islets having a diameter of about 100 μm were hand-picked into prepared wells using a p100 or p200 micropipette using a digital desktop microscope (10 times). Similarly, 10 capsules with islets of similar size were added to the prepared wells in triplicate. Once all samples were added to the appropriate wells, the culture medium was replaced with 1 mL of warm 2.8 mM glucose KRBH buffer. As described above, images of free and encapsulated islets were taken using a conventional bright field microscope for IEQ calculation. The 24-well plates were incubated in a humidified incubator at 37 ° C and 5% CO2 for 1 hour. Replace the 2.8 mM glucose KRBH buffer with fresh 1 mL of 2.8 mM glucose-KRBH buffer and incubate for 1 hour as before. Collect the 2.8 mM glucose KRBH buffer into a labeled 1.5 mL microcentrifuge tube as a baseline for islet insulin secretion measurements. Add 1 mL of warm 16.7 mM glucose KRBH buffer to free and encapsulated islets and incubate for 1 hour as before. 16.7 mM glucose KRBH buffer is used for insulin secretion from islets after stimulation. Samples were stored at -20 °C until further use.

[0172] Insulin secretion was measured by Ultra-sensitive rat insulin ELISA kit from Crystal Chem (Downers Grove, IL, USA) and normalized to the IEQ of each well.

[0173] result

[0174] like Figure 8 As shown, explanted GSIS-islets were functional in two out of three rats (healthy rats receiving islets) after short-term (2 weeks) transplantation. Figures 9A-9B Shown is a 2-week live / dead staining on explanted TEC-cy columns containing rat pancreatic islets transplanted into healthy Wistar rats. Long-term GSIS data (42 days) of retrieved explants are shown. Figures 10A-10B Short-term transplants (conditions labeled Ahr-1 and Ahr-3, 14 days) were compared to long-term (42 days) conditions labeled Ahr-4, Ahr-5, and Ahr-6. Long-term explants were also imaged, as shown. Figures 11A-11B BG data were tracked over a period of one month in diabetic mice transplanted with TEC-cy columns at three islet loading densities (human islets) and TEC-cy. Fig.12 ).from Fig.12 It can be seen that the transplantation effect of encapsulated islets is better than that of free islets and blank control. Fig.13 Shown are the BGs of diabetic mice 29 days after treatment with TEC-cy capsules containing human islets. Figures 14A-14F Live / dead staining images of human islets explanted in TEC-cy capsules after 29 days in diabetic mice are shown. Figures 14A-14F It can be seen that the islets are still alive.

[0175] Pericapsular fibrosis (PCO) was assessed from capsules explanted from mice. All capsules retrieved from the explantation procedure were transferred to microscope slides and imaged using a Nikon Eclipse Ti inverted microscope. An automated stage was used for software-controlled stage translation to capture brightfield images, which were stitched using Nikon NIS Elements AR5.11.01 software to form composite images. For PCO analysis, individual capsules in the composite images were manually assessed according to PCO scoring categories 1, 2, 3, and 4, corresponding to 0-24%, 25-49%, 50-74%, and 75-100% hyperplastic capsule coverage, respectively. It should be noted that the analysis was limited to one side of the capsule being assessed.

[0176] Formation of TEC-cy hydrogel patches (blank)

[0177] To prepare TEC-cy hydrogel patches, TEC-cy gel-forming solution was prepared as described, and 1-3 wt% alginate was loaded into the syringe of Advanced Solutions BioBot Basic and equipped with a 0.5” 20G blunt-tip needle. FRESH TM (Free-form reversible embedding of suspended hydrogels) The support bath was prepared with 9 mM calcium chloride. TM The bath acts as a Bingham plastomer during the printing process, and the 9mM calcium chloride causes partial ionic crosslinking of the alginate during the printing process, providing sufficient stiffness for the newly formed tubular segments. It is worth noting that the 9mM calcium concentration is low enough to allow further crosslinking of the interconnecting layers within the final three-dimensional structure. Then, 10-15mL of this FRESH containing 9mM calcium chloride is added. TM The support bath is placed into a single well of a 6-well plate. The predetermined patch shape is printed in a cross-hatched pattern with an inter-filament distance ranging from 250 μm to 1500 μm, preferably 400 μm to 700 μm. The linear printing speed is matched to the flow rate (which in turn is related to the applied pressure and the rheological properties of the bio-ink for this printer model) to obtain the target filament diameter. After printing is completed, the 6-well plate is placed in an incubator at 37°C for 0.5-1 hour to allow FRESH TMThe support bath was completely liquefied. The patch was then removed and placed in a well of a 6-well plate with a gel bath (pH 7.4) consisting of 100 mM calcium chloride, 35 mM HEPES, and 0.45 wt.% sodium chloride for a few minutes, followed by a saline rinse. In other cases, a barium gelling bath (pH 7.4) containing 10 mM barium chloride, 35 mM HEPES, and 0.45 wt.% sodium chloride was used for 5 minutes. Fig.15 The resulting three-layer TEC-cy patch formed from 3 wt. % alginate and cured with calcium chloride is shown.

[0178] Example 8: Encapsulation of human donor islets in TEC-cy patches

[0179] For bioprinted patches with human donor islets, TEC-cy gel-forming solution was prepared as described, except that the concentration of polymer in HBS was 1.2-fold higher and the amount of alginate used was 2.4 wt.%. The resulting solution was sterilized using a 0.22 μm syringe filter in the BSC. The solution was carefully diluted 5:1 with human donor islets in HBS to obtain a printing solution containing a polymer concentration equal to the previously described TEC-cy gel-forming solution with a 2 wt.% alginate content and a final filament length of islets >80 IEQ / cm. This solution was loaded into the assembled syringe of an Advanced Solutions BioBot Basic placed in the same BSC and equipped with a 0.5" 20G blunt-tip needle. FRESH TM The support bath was prepared in sterile form by soaking in 70% ethanol for 1 hour and then purified by subsequent repeated washing and centrifugation to a calcium chloride content of 9 mM. 10-15 mL of this fresh calcium chloride-enriched solution was added to the TM The support bath was placed in a single well of a 6-well plate. Four layers of 2 x 2 cm patches were printed in a cross-hatched pattern with an inter-filament distance of 750 μm (although there were offset layers). The printing speed was matched to the flow rate (which is related to the applied pressure and the rheological properties of the bioink for that printer model) to obtain a filament diameter that matched the filament diameter required for printing a given patch and to form a four-layer patch in less than 3 minutes. Multiple of these patches were printed with the same ink and the resulting patches were cultured for up to 4 days. Patches were selected at days 0, 1, 2, and 4, stained with phenanthridin / calcein AM live / dead staining, and evaluated by confocal microscopy to observe the extent to which islet viability was maintained over time. The functionality of the patches was also assessed by quantifying insulin release in response to a glucose-stimulated insulin secretion (GSIS) experiment.

[0180] Confocal images of live / dead stained islet clusters in bioprinted TEC-cy patches containing 2 wt.% alginate showed that islets could be printed at high concentrations (>80 IEQ / cm of filament length) while maintaining cluster viability and keeping them away from the edge of the printed filament over 7 days ( Figures 16A-16B ).

Claims

1. A hydrogel column, comprising: Thiol-ene cross-linked polymers comprising a first side-chain functionalized backbone polymer functionalized with an activated olefin that cross-links with free or protected thiol-containing groups present on a second side-chain functionalized backbone polymer; and A biomaterial encapsulated in the thiol-ene cross-linked polymer.

2. The hydrogel column according to claim 1, wherein the hydrogel column has an aspect ratio of at least 5, preferably at least 100.

3. The hydrogel column according to claim 1 or 2, wherein the hydrogel column further comprises alginate, hyaluronic acid, gelatin, hydroxypropyl cellulose, carboxymethyl cellulose, methyl cellulose or elastin.

4. The hydrogel column according to any one of claims 1 to 3, wherein the biomaterial is a cell, a cell aggregate or a cell spheroid, and the hydrogel column optionally further encapsulates angiogenic agents and / or chemotactic agents.

5. The hydrogel column according to any one of claims 1 to 4, wherein the activated olefin is vinyl sulfone, maleimide, acrylate or methacrylate.

6. The hydrogel column according to any one of claims 1 to 5, wherein the thiol-containing group is 2-pyridinethiol or cystamine.

7. The hydrogel column according to any one of claims 1 to 6, wherein the backbone polymer is a homopolymer of polyacrylic acid, a homopolymer of polymethacrylic acid, or a copolymer of acrylic acid and methacrylic acid.

8. The hydrogel column according to claim 5, further comprising a capping agent for neutralizing vinyl sulfone groups on the surface of the hydrogel column.

9. The hydrogel column according to any one of claims 1 to 8, wherein the hydrogel column has an outer diameter of less than 2000 μm, preferably less than 1000 μm, and more preferably less than 600 μm.

10. The hydrogel column according to any one of claims 1 to 9, wherein the thiol-ene cross-linked polymer forms a shell encapsulating a core of the biomaterial.

11. The hydrogel column of any one of claims 1 to 10, wherein the shell has a non-uniform density, wherein the outer surface has a higher density than the inner surface as measured by fluorescence microscopy.

12. A three-dimensional hydrogel structure formed by interconnected hydrogel tubes as defined in any one of claims 1 to 11, comprising: A plurality of the thiol-ene cross-linked polymers form at least a portion of the hydrogel columns connected to each other, wherein each of the hydrogel columns is connected by thiol-ene cross-links forming a continuous cross-linked structure.

13. A three-dimensional hydrogel structure according to claim 12, wherein the three-dimensional hydrogel structure is a patch formed by 3D printing the hydrogel columns into a shape, wherein the hydrogel columns or portions thereof intersect or cross each other to form a two-dimensional array of micropores, and the two-dimensional array of micropores is preferably designed to maximize the surface area required for metabolic exchange of therapeutic cells, preferably wherein the biomaterial is able to migrate between the intersecting columns or portions thereof.

14. A method for producing a hydrogel column, comprising: Continuously extruding or co-extruding a first polymer containing free or protected thiol groups and a second polymer containing vinyl groups into a bath containing reactants, preferably a water bath, to drive gelation of the first polymer and the second polymer; and A cross-linking reaction is caused between the thiol group and the vinyl group.

15. A method for producing interconnected hydrogel columns, comprising: extruding or co-extruding a composition comprising a first polymer containing free or protected thiol groups and a second polymer containing vinyl groups into a bath containing reactants, preferably a water bath, to drive gelation of the first polymer and the second polymer, and wherein the bath has a low concentration of reactants such that the thiol-ene polymers are only partially cross-linked to form a plurality of polymer pillars; and forming a desired shape with the plurality of polymeric columns; as well as The plurality of polymer pillars are exposed to a reducing agent to further cross-link the plurality of polymer pillars to form the interconnected hydrogel pillars.

16. The method of claim 14 or 15, wherein the extruding or coextruding further comprises extruding or coextruding a biocompatible polymer with the first polymer and the second polymer, the biocompatible polymer being selected from the group consisting of alginate, hyaluronic acid, gelatin, hydroxypropyl cellulose, carboxymethyl cellulose, methyl cellulose, or elastin.

17. A method according to any one of claims 14 to 16, wherein the biocompatible polymer is alginate and the bath comprises ions selected from the group consisting of calcium, barium, strontium, copper, zinc, manganese, cobalt, lead, iron or aluminium, preferably the ions are present in a concentration of 5 mM to 100 mM.

18. The method according to any one of claims 14 to 17, wherein the reactant is a reducing agent, preferably tris(2-carboxyethyl)phosphine (TCEP) or tris(hydroxypropyl)phosphine (THPP), and preferably the reactant is present in a concentration of 5 mM to 100 mM.

19. A method according to any one of claims 14 to 18, further comprising providing an introduced capping agent prior to the final wash to convert residual vinyl sulfone groups to more biocompatible groups.

20. The process according to any one of claims 14 to 19, wherein the ratio of thiol groups to vinyl(enyl) groups is from 0.95:1.05 to 0.65:1.35, preferably from 0.9:1.1 to 0.5:1.5, and most preferably from 0.8:1.2 to 0.6:1.

4.

21. A method according to any one of claims 14 to 20, comprising extruding or co-extruding the polymer into the bath through a needle, preferably a blunt-ended syringe.

22. The method of any one of claims 14 to 21, wherein the thiol groups of the first polymer are protected thiol groups, and preferably the first polymer is poly(methyl vinyl ether-alt-maleic anhydride) bearing the protected thiol groups.

23. A transplant comprising at least one hydrogel column as defined in any one of claims 1 to 11 and optionally a supporting substrate, wherein at least one of the hydrogel columns is substantially retrievable from a transplant site in a living mammal after a transplant period of six weeks to one year or more, such as 2-3 years.

24. A method of transplanting the transplant of claim 23, comprising: placing the graft on expanded omental tissue of a subject in need thereof; folding the omental tissue to form a pocket containing the graft; as well as The omentum tissue is fixed during pocket formation by suturing and / or using tissue glue, preferably wherein the tissue glue comprises a mixture of fibrinogen and thrombin, or the like.

25. The method of claim 24, wherein the method is an open surgery, a laparoscopic procedure.

26. A hydrogel comprising a first side chain functionalized backbone polymer functionalized with an activated olefin cross-linked with cystamine containing groups present on a second side chain functionalized backbone polymer.

27. The hydrogel of claim 26, further comprising alginate, hyaluronic acid, gelatin, hydroxypropyl cellulose, carboxymethyl cellulose, methyl cellulose, or elastin.

28. The hydrogel according to claim 26 or 27, wherein the hydrogel encapsulates biological material of mammalian or bacterial origin, preferably cells, cell aggregates or cell spheroids.

29. The hydrogel of any one of claims 26 to 28, wherein the activated olefin is a vinyl sulfone, an acrylate or a methacrylate.

30. The hydrogel of any one of claims 26 to 29, wherein the backbone polymer is a homopolymer of polyacrylic acid, a homopolymer of polymethacrylic acid, or a copolymer of acrylic acid and methacrylic acid.

31. The hydrogel of any one of claims 26 to 30, wherein the cystamine-containing groups have a poly(methyl vinyl ether-alt-maleic anhydride) backbone polymer.

32. The hydrogel of any one of claims 26 to 31, further comprising a capping agent that neutralizes the vinyl sulfone groups on the surface of the hydrogel column.

33. The hydrogel of any one of claims 26 to 32, wherein the hydrogel is a capsule, a column or a patch.

34. A kit for encapsulating a biological material, comprising: A solution containing 5 mol.% to 35 mol.% of a polymer containing free or protected thiol groups; A solution containing 5 mol.% to 35 mol.% of a vinyl-containing polymer; alginate in a buffer solution at a concentration of 1 wt.% to 4 wt.% suitable for receiving the biomaterial to be encapsulated; Optionally, a calcium or barium salt to form an ionic gel bath; and Optionally, a reducing agent for the ion bath or for post-printing gelation of the thiol-ene polymer.

35. Use of the kit according to claim 34 for producing a hydrogel column as defined in any one of claims 1 to 11.

36. Use of the kit according to claim 34 for producing the three-dimensional hydrogel structure defined in claim 12 or 13.

37. Use of the kit of claim 34 in a method for producing a hydrogel column or interconnected hydrogel columns as defined in any one of claims 14 to 22.

38. Use of the kit according to claim 34 for producing the transplant as defined in claim 23.

39. Use of the kit of claim 34 for producing a hydrogel as defined in any one of claims 26 to 33.

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