Crosslinked, modified and hydrolyzed methacrylic anhydride microgels for biomaterials applications

Abstract

There is provided microparticles crosslinked with disulfide bonds and methods of producing the same. The microparticles are particularly useful for the cryopreservation of cells. The disulfide bonds are provided from a functionalization of methacrylic anhydride based microparticles with cystamine or from using methacrylic anhydride based monomers containing a cystamine link core with the disulfide bond, and beneficially by combining both modes of disulfide crosslinking. Other uses for these microspheres include as printing support materials (jammed gels) for 3D printing hydrogels, optionally containing therapeutic cells.

Description

[0001] MICROGELS FOR BIOMATERIALS APPLICATIONS

[0002] CROSS-REFERENCE TO A RELATED APPLICATION

[0003] [1] This disclosure claims priority from U.S. provisional application number 63 / 575,203 filed on April 5, 2024, which is incorporated herein by reference in its entirety.

[0004] TECHNICAL FIELD

[0005] [2] This disclosure relates to the field of microgels, particularly microgels useful in the formation of jammed gels for use in cryopreservation of biological materials such as cells and cell clusters, printing support baths for 3D bioprinting of cells and cell clusters in gel-forming polymer suspensions, and as a cell suspending medium for cells and cell clusters.

[0006] BACKGROUND OF THE ART

[0007] [3] Hydrogels have long been recognized as useful materials to interface with cells and tissues as it is recognized that hydrogels can mimic certain properties of natural tissue, and can hence be thought of as synthetic extracellular matrix (ECM) materials. Examples range from synthetic crosslinked hydrogels based on hydroxyethyl methacrylate (HEMA) used as contact lenses, to Matrigel™, a commercially available ECM derived from mouse cancer cell-lines that allows culturing stem cells, and many forms of polyethylene glycol-based hydrogels.

[0008] [4] Microgels have been used as cryoprotective materials for cryostorage of mammalian cells. Cell attachment within matrices constructed of inverse suspension microgels with broad particle size distribution has been shown.

[0009] [5] Improvements in microgel compositions are still desired, for better long-term cryostorage, cell compatibility and cell survivability, among other aspects.

[0010] SUMMARY

[0011] [6] In one aspect, there is provided a method for producing a microparticle, the method comprising: heating a marginal solvent (single or mixed) containing dissolved methacrylic anhydride monomers to polymerize the monomers and obtain a microparticle; crosslinking the microparticle with cystamine; reacting the microparticle with a compound comprising a primary amine group and a tertiary amine group, and hydrolyzing any residual anhydride groups on the microparticle by contacting the microparticle with an alkaline aqueous solution. [7] In a further aspect, there is provided a method of producing a microparticle, the method comprising: heating a marginal solvent comprising methacrylic anhydride together with one or more of a crosslinker of formula (1) and / or (2), or a compound selected from compounds A, B, C, D, E, F, G, H, I, J, and combinations thereof, and a bis (meth)acryloyl or bis or tetra(meth)acrylamide derivative thereof and obtain a microparticle; converting the majority of the remaining anhydride groups by reacting the microparticle with an excess of a compound comprising a primary amine group and a tertiary amine group such as dimethylaminopropylamine (DMAPA), and hydrolyzing any residual anhydride groups on the microparticle by contacting the microparticle with an alkaline aqueous solution. Optionally, the method further comprises in between steps i) and ii), a step of crosslinking the microparticle with cystamine. wherein x and y are each independently an integer from 0 to 7;

[0012] [8] In a further aspect, there is provided a method to introduce additional beneficial molecular functions into the microgels, by reacting some of the anhydride group with molecules designed to form binary complexes by hydrogen bonding, such as 1-(6-aminohexyl)-3-(6-methyl- 4-oxo-1 ,4-dihydropyrimidin-2-yl)urea (UPy) or analogous hydrogen-bonding groups like nitrogenous bases (uracil, thymine, etc), 2,7-diamido-1,8-naphthyridine (DAN), diacyldiaminopyridine (DAP), ureidoguanosine (UG), cyanuric acid (CA), dopamine, RGD and other peptide sequences, and others. This is done by introducing amine-functional UPy before modifications with cystamine, and the molecule containing a primary and tertiary amine, or concurrently with one or both of these, or between modifications with these two molecules. Introduction of UPy causes moderate association between the microgels, with the strength of association increasing with the degree of modification with UPy and increasing with lower temperature, features that have benefits for use of these microgels in 3D printing baths as they allow manipulation of printing bath viscosity through degree of UPy functionalization, as well as through temperature of the printing bath. Furthermore, melting of the thermally labile complexation between UPy-modified microgels, optionally with the help of hydrogen-bonding-breaking molecules such as urea, may help facilitate removal of the microgels from the formed 3D hydrogel.

[0013] [9] The resulting microparticles are degradable both in vivo, as well as by exposure to millimolar concentrations 0.1 - 50 mM, 0.1 - 10 mM, 1 - 10 mM, 1 - 5 mM, or 1 - 3 mM (3.18x10’3- 1.6 molar eq., 0.03 - 0.16 molar eq., or 0.03 - 0.09 molar eq. of reducing agent to disulfide linkages when used in a 1 :2 TCEP:Microgel suspension volume ratio)) of commonly available, and cell-compatible reducing agents such as tris(2-carboxyethyl)phosphine (TCEP), tris(3- hydroxypropyl)phosphine (THPP), or glutathione. This feature allows these microgels to be more easily removed after use as cryoprotective jammed gels. This degradability also can facilitate the removal after use of these jammed gels as transparent shear-thinning supporting baths for 3D printing, e.g. 3D bioprinting of high surface area hydrogel constructs or patches containing therapeutic cells, for use in human therapies for, e.g., T1 D.

[0014]

[0010] In some embodiments, the marginal solvent is selected from heptane, toluene, xylenes, methyl ethyl ketone (MEK), tetrahydrofuran (THF), acetonitrile, ethyl acetate, benzene, N,N-dimethylformamide (DMF), 1 ,4-dioxane, acetone, cyclohexane, or mixtures thereof.

[0015]

[0011] In some embodiments, the heating used to form the microparticles is performed at a temperature of from 20 to 85 °C, 30 to 85 °C, or 45 to 60 °C, or 50 to 55 °C.

[0016]

[0012] In some embodiments, the marginal solvent further comprises a polymerization initiator, for example a thermally or photochemically activated free radical polymerization initiator.

[0013] In some embodiments, the polymerization initiator is 2,2'-azobis(2,4 dimethylvaleronitrile) (ADVN).

[0017]

[0014] In some embodiments, the polymerization is carried out at temperatures between 0 and 40 °C, with the help of photo-initiation, e.g. by shining a LIV light into the reaction mixture containing photo initiator, and example of which is AIBN.

[0018]

[0015] In some embodiments, the microparticle is crosslinked by disulfide bonds. The disulfide bond may be from either cystamine, or one or more of the modified methacrylic anhydride monomer of formula 1 or 2, or a mixture of cystamine and (1 and / or 2).

[0019]

[0016] In some embodiments, the microgels can also degrade spontaneously in absence of TCEP, due to processes that may include disulfide interchange and hydrolysis. Reductive cleavage and hydrolysis may also beneficially combine to reduce the concentration of TCEP required.

[0020]

[0017] In yet a further aspect, there is provided a method of cryopreserving cells comprising: providing microparticles as described herein or as obtained by the methods described herein; contacting the cells with the microparticles; and freezing the cells.

[0021]

[0018] In another aspect, the cells may be individual cells, or may be cell clusters comprising a multiplicity of cells, with the number of cells per cluster ranging from 1 to 5000, preferably 2 to 2000, and most preferably 10 to 300 cells. In another aspect, the cells may form a cell-sheet of unlimited extension in two dimensions, and a third dimension (thickness) in the range of 1 to 20, and preferably 1 to 5 cells.

[0022]

[0019] In an additional aspect, there is provided a method of cryopreserving cells comprising combining the microparticle as described herein or as obtained by the methods described herein in an aqueous suspension in a microparticle to cell volume ratio of 10000:1 to 1 :1 , preferably 5000:1 to 5:1 , and most preferably 500:1 to 10:1 , and freezing the suspension of microparticles and cells preferably to about - 80 degrees centigrade, or at least - 60 degrees centrigrade. This freezing process can follow usual steps in the art such as controlled freezing at approximately 1 degrees centigrade per minute, as obtained using a Mr. Frosty™ or other technology developed for this purpose and familiar to those skilled in the art. There is also provided the use of liquid nitrogen for long term storage of the microparticles and cell cryopreservation.

[0020] In a further aspect, a cell cryopreservative comprising: a population of polymeric microparticles comprising a reaction product of a polymerization comprising a structure

[0023] and a disulfide crosslinker, where additionally the anhydride groups have been converted by reaction with dimethylaminopropylamine or other analogous molecules comprising a primary amine and a tertiary amine, so as to form polyampholytes. In some embodiments, the polyampholytes are near-stoichiometric charge ratios, however other stoichiometric ratios are also possible such as a 40:60 to 60:40 ratio of cationic: anionic ratio. The ratio can vary from 10:90 to 90:10, from 20:80 to 80:20 or from 30:70 to 70:30.

[0024]

[0021] In some embodiments, polymerized microparticles crosslinked using formula (1) or formula (2), or a combination of cystamine with (1 and / or 2), where additionally anhydride groups are reacted with N2-(2-aminoethyl)-N1 ,N1 -dimethylethylenediamine or other molecules that are integrated by at least one primary amine group and at least one tertiary amine group that introduce one or more cationic charges to the particle, such cationic particles. These are useful as carriers for antigens.

[0025]

[0022] In still an additional aspect, there is provided a cell cryopreservative comprising a microparticle formed by disulfide bond crosslinks and a solvent that is supportive of cryopreservation. In some embodiments, this solvent is an aqueous media containing essential nutrients for cells, as well as optionally one or more apoptosis inhibitors, as is common in the art. This solvent may also incorporate a small amount of conventional cryoprotective molecules such as dimethylsulfoxide (DMSO) in amounts of up to 2 vol%, hydroxy-containing cryoprotective agents such as propylene glycol, at levels up to 10 vol%, betaine at levels up to 10 wt%.

[0023] In some embodiments, preferably, at least 90%, at least 95%, at least 98%, at least 99% of the crosslinks formed between backbone chains in the microparticle after hydrolysis of the anhydride crosslinkes are the disulfide bond crosslinks. In some embodiments, the microgels are significantly swollen in the cryosolvent such that they expand to leave no interstitial free volume between the microparticles. In some embodiments, the interstitial volume is of less than 5%, less than 2%, less than 1%, less than 0.5% or less than 0.1%. In one embodiment, this includes 1 to 20 wt%, and preferably 4 to 10 wt% of microgels being swollen in the cryosolvent, to form a jammed gel (dense packing of swollen microgel particles) that can physically support cells and prevent sedimentation and aggregation.

[0026]

[0024] In an additional aspect, jammed gels such as those described above may also serve as printing support baths for 3D bioprinting. In this application, therapeutic cells embedded in gel formers may be extruded using a 3D printer, into a jammed gel of degradable microgel particles, to form a hydrogel device containing therapeutic cells (for example having a high surface area). One example of such a device is a patch, consisting of continuous strings of hydrogel, extruded in such a way so as to form a pattern of string segments. The gel formers are for example selected from alginate, pluronic, hyaluronic acid, gelatin, hydroxypropylcellulose, carboxymethylcellulose, methylcellulose or elastin, and may optionally contain synthetic polymers capable of spontaneous thermal, ionic or covalent gelation during extrusion, or analogous induced gelation post-extrusion, or a combination of both of these. In these cases, 3D structures can be generated while being supported by the jammed gel, which allows for 3D printing of materials that otherwise would not initially be strong enough to support themselves lose their shape fidelity upon extrusion in the absence of a supporting bath. Each layer of string segments may be one long string segment curved appropriately to form the overall pattern in the layer. As well, the whole device may be a single continuous string that is not interrupted even at the printing transitions from one layer to the next layer. Each layer, and the whole assembly, may be flat, or assume a three dimensional shape, accessible using current 3D printers.

[0027]

[0025] Many further features and combinations thereof concerning the present improvements will appear to those skilled in the art following a reading of the instant disclosure.

[0028] BRIEF DESCRIPTION OF THE DRAWINGS

[0029]

[0026] FIG. 1A is a microscopy image of microparticles obtained in Example 1.

[0030]

[0027] FIG. 1B is a close-up of Fig. 1A.

[0028] FIG. 2A is a fluorescent microscopy image of the microparticles of Example 1 functionalized with a fluorescent agent.

[0031]

[0029] FIG. 2B is a close-up of Fig. 2A.

[0032]

[0030] FIG. 3A is a microscopy image of the microparticles obtained in Example 2.

[0033]

[0031] FIG. 3B is a close-up of Fig. 3A.

[0034]

[0032] FIG. 4A is a microscopy image of the frozen microparticles in Example 2.

[0035]

[0033] FIG. 4B is a close-up of Fig. 4A.

[0036]

[0034] FIG. 5 is an optical Micrograph imagey of microparticles modified with 1-(6- aminohexyl)-3-(6-methyl-4-oxo-1,4-dihydropyrimidin-2-yl)urea (UPy).

[0037]

[0035] FIG. 6 is a micrography of microparticles modified with dopamine.

[0038]

[0036] FIG. 7 is a micrography of microparticles modified with RGD.

[0039]

[0037] FIG. 8A is a fluorescent microscopy image showing the fibroblast cell viability (negative control condition).

[0040]

[0038] FIG. 8B is a fluorescent microscopy image showing the fibroblast cell viability after thawing from being frozen in the microgel in the absence of dimethylsufloxide.

[0041]

[0039] FIG. 8C is a fluorescent microscopy image showing the fibroblast cell viability after thawing from being frozen in the microgel in the presence of dimethylsufloxide.

[0042]

[0040] FIG. 8D is a fluorescent microscopy image showing the fibroblast cell viability (positive control condition).

[0043]

[0041] FIG. 9A is a fluorescent microscopy image of live dead staining for induced pluripotent stem cells (iPSC) (negative control).

[0044]

[0042] FIG. 9B is a fluorescent microscopy image of live dead staining for iPSC (positive control).

[0045]

[0043] FIG. 9C is a fluorescent microscopy image of live dead staining for iPSC (solution I).

[0044] FIG. 9D is a fluorescent microscopy image of live dead staining for iPSC (solution II).

[0046]

[0045] FIG. 10A is a fluorescent microscopy image of human mesenchymal stromal cells frozen with 2% DMSO.

[0047]

[0046] FIG. 10B is a fluorescent microscopy image of human mesenchymal stromal cells frozen with 0% DMSO.

[0048]

[0047] FIG. 10C is a fluorescent microscopy image of human mesenchymal stromal cells frozen in a microgel with 2% DMSO.

[0049]

[0048] FIG. 10D is a fluorescent microscopy image of human mesenchymal stromal cells frozen in a microgel with 0% DMSO.

[0050]

[0049] FIG. 11 A is a fluorescent microscopy image of a human donor islets (positive control).

[0051]

[0050] FIG. 11B is a fluorescent microscopy image of a human donor islets (in microgel).

[0052]

[0051] FIG. 12 is a fluorescent microscopy image of hybrid clusters of human induced pluripotent stem cells.

[0053]

[0052] FIG. 13 is a fluorescent microscopy image of human donor islets suspended in microgels.

[0054]

[0053] FIG. 14 is a micrography of anionic microgel modified with 750 Da molecular weight

[0055] PEG.

[0056]

[0054] FIG. 15A shows a series of close up, focussed images of an intersection of a patch printed in microgels.

[0057]

[0055] FIG. 15B is a stitched image of an entire patch printed in microgels.

[0058] DETAILED DESCRIPTION

[0059]

[0056] The term “microgel” as used herein refers to lightly crosslinked polymer systems in the form of microparticles that are swollen by a solvent. The term “hydrogel” as used herein refers to lightly crosslinked polymer systems that are swollen in water and related aqueous cell media as may be appropriate for use with mammalian and other cells.

[0057] The term “microparticle”, while generally used to refer to particles between 1 and 1000 pm in size, as used herein can also encompass, unless the context dictates otherwise, submicron particles in the 0.2 to 1 pm size (i.e., nanoparticles within this size range). In preferred embodiments, the microparticles have a particle diameter between 0.2 and 50 pm, more preferably between 0.3 and 30 pm, or still more preferably between 1 and 20 pm.

[0060]

[0058] The term “covalently crosslinked” as used herein with respect to a polymer matrix refers to the formation of covalent bonds between polymer chains that hold together the polymer matrix, a microparticle in this work. It is not possible for the microparticle to undergo facile dissolution into individual polymer chains when the covalent crosslinks are present. Covalent crosslinks here include the sulfur-sulfur bonds (disulfide bonds) present in cystamine as well as methacrylic anhydride and the methacrylic crosslinkers (1) and (2).

[0061]

[0059] The term “fusion” as used herein refers to the permanent covalent interconnection between string segments in adjacent layers at their “cross-over points” where string segments overlap.

[0062]

[0060] The term “biocompatible” as defined herein refers to compounds or microparticles that are compatible with in vitro or in vivo prolonged contact with cells and / or specific biological tissues. Biocompatible compounds or microparticles do not elicit a significant negative effect on the cell survivability, cell function, and / or tissue function, whereby biocompatibility is usually specified in terms of being compatible with a particular tissue or cell environment.

[0063]

[0061] The term “jammed” as used herein in the context of gels and microgels, refers to close- packed microgel particles that form a shear-thinning medium that appears solid at rest but can be temporarily mobilized by moderate shear forces.

[0064]

[0062] Accordingly, there is provided the fabrication of microgel particles that have desirable cryopreservative properties for biological materials, preferably a cell, a cell cluster, a tissue or a cell spheroid, of either mammalian or bacterial origin. The microgels contemplated herein are biocompatible. They are also biodegradable so as to enable, for example, infusion of thawed cryostored therapeutic cells, or implantation of 3D printed (and optionally cryostored) cellcontaining hydrogel patches, without need for complete removal of the microgels. These microgel particles are produced in two steps. First, a polymerization with methacrylic anhydride is performed to obtain microparticles. The polymerization can be performed in a marginal solvent which may be organic (for example heptane, toluene, xylenes, methyl ethyl ketone (MEK), tetrahydrofuran (THF), acetonitrile, ethyl acetate, benzene, cyclohexane, or mixtures thereof), optionally under heating (for example at a temperature of from 20 to 85 °C, from 20 to 70 °C, 40 - 65 °C, and preferably 50-55 °C) or photo-initiation at similar or lower temperature, and in the presence of a polymerization initiator such as 2,2’-Azobis-dimethylvaleronitrile (ADVN) or 2,2’- Azobis-isobutyronitrile (Al BN) or other initiators or initiation methods known to those skilled in the art. (methacrylic anhydride).

[0065]

[0063] After polymerization, the microgels are functionalized by reaction with a limited amount of cystamine to generate crosslinks comprising disulfide bridges. Subsequently, the remaining anhydrides are reacted with an excess of a compound containing a primary and a tertiary amine group such as dimethylaminopropylamine (DMAPA), and then any residual anhydride groups are hydrolyzed by bringing the microgels into contact with an aqueous, preferably alkaline, solution. The microgel particles obtained after hydrolysis are only crosslinked with the disulfide bonds from the cystamine. (cystamine)

[0066]

[0064] The microparticles are degradable both in vivo, as well as by exposure to millimolar concentrations 0.1 - 50 mM, 1 - 10 mM, 1 - 5 mM, or 1 - 3 mM (3.18x10-3- 1.6 molar eq., 0.03 - 0.16 molar eq., or 0.03 - 0.09 molar eq. of reducing agent to disulfide linkages) of commonly available, and cell-compatible reducing agents such as tris(2-carboxyethyl)phosphine (TCEP), tris(3-hydroxypropyl)phosphine (THPP) or glutathione. This feature allows these microgels to be more easily removed after use as cryoprotective jammed gels. This degradability also can facilitate the removal after use of these jammed gels as transparent shear-thinning supporting baths for 3D printing, e.g. 3D bioprinting of high surface area hydrogel constructs or patches containing therapeutic cells, for use in human therapies for, e.g., T1 D.

[0065] As well, natural reducing agents such as glutathione or ascorbic acid present in tissue can also reduce such disulfide bonds and facilitate in-vivo degradation of any microgels not completely degraded in vitro prior to infusion or implantation of the therapeutic cell load.

[0067]

[0066] In addition, the methacrylic anhydride can be modified to incorporate the disulfide bonds and the cystamine structure in between the methacrylate terminal groups. Accordingly, a polymerization can be performed with methacrylic anhydride and also with the modified methacrylic anhydrides incorporating disulfide bridges. In such embodiments, the second step of functionalization with cystamine becomes optional because some disulfide crosslinks are already incorporated during polymerization, but can be beneficial as it allows greater control over placement of the disulfide crosslinker within the microgel. One may even, in some embodiments, mix cystamine with methacrylic anhydride prior to or during the copolymerization and carrying out formation of (2) just prior to or during the precipitation polymerization. The modified methacrylic anhydride can for example be:

[0068]

[0067] The resulting microparticles have the following structure respectively based on cystamine, formula 1 or formula 2.

[0069]

[0070]

[0071]

[0068] “x”, “y” and “z” are repeating subunits. For simplicity, each group is shown as separately repeating to form block copolymer structures, however, the groups shown can be alternately repeating or have a random sequence along the backbone.

[0072]

[0069] In some embodiments the compound containing a primary and a tertiary amine group has the formula NH2-R-N(R)2 where each R is independently a C1-C6, C1-C5, C1-C4, or C1-C3 alkyl that are each independently optionally substituted, optionally interrupted, and / or optionally terminated by S, O or N. In some embodiments the formula is NH2-R-N(R2)2 where R2is methyl or ethyl and preferably methyl, and R is as previously defined. The compound has one primary amine to react with residual anhydride to form an amide bond and in that process generate a carboxylic anion, and (at least one and usually one) tertiary amine that does not react with anhydride but will contribute a quaternary ammonium cation to balance the above carboxylic anion, thus forming a charge-balanced, polyampholytic microgel.

[0073]

[0070] These microparticles are usually still further subjected to a crosslinking postfunctionalization with cystamine. The combination of two types of crosslinking processes, namely (a) crosslinking through the divinyl crosslinkers (1) and (2) that takes place during the initial precipitation polymerization, and (b) post-crosslinking of the initially formed copolymer microparticles by reaction with cystamine, convey certain advantages. These include better control over the actual placement of the crosslink groups, and faster degradation using reducing agents such as TCEP, THPP and glutathione. Specifically, post-functionalization with cystamine places the resulting crosslinks close to the surface of the microparticles, while incorporation of either or both of divinyl crosslinker (1) or (2) leads to crosslinking taking place predominantly during the initial phases of the precipitation polymerization which in turns leads to preferential crosslinking of the microparticle cores. A proper combination of both crosslinking methods and molecules allows forming microparticles with balanced crosslinking throughout the microparticle, or other distributions as may be deemed beneficial for the function of the microparticles, either with regard to use in printing baths or cryo media, and / or with regards to their ultimate degradation using reducing agents.

[0074]

[0071] In some cases it may be beneficial to modify the as-formed particles, either before or after post-modification with cystamine, by introduction of a hydrogen-bonding moiety called UPy or analogous hydrogen-bonding groups like nitrogenous bases (uracil, thymine, etc), 2,7-diamido- 1 ,8-naphthyridine (DAN), diacyldiaminopyridine (DAP), ureidoguanosine (UG), cyanuric acid (CA), dopamine and others. These moieties have the ability to form binary hydrogen-bonded complexes involving two to four hydrogen bonds per complex, operating in cooperative fashion. This complexation may enhance the cohesive interaction between microgels during 3D printing, which can prove beneficial for the surface quality and overall integrity of the hydrogel being printed. The hydrogen bonds are thermally labile, and may be designed to melt at higher temperatures (e.g. at more than 70, 75, 80 or 85 °C) to facilitate removal of the printed hydrogel from the microgels bath. This microgel cohesion is also dependant on UPy loading, and UPy distribution within the microgels and in particular at the surface. Amounts of UPy to be introduced may vary from 0.1 to 20 mol% of the originally present anhydride groups, and preferably between 1 and 10 mol%.

[0075]

[0072] In some embodiments, the method leverages a post-functionalization step with excess of the compound containing a primary and tertiary amine group such as dimethylaminopropylamine (DMAPA) to convert most of the remaining anhydride groups into a charge-balanced group comprising one carboxylic acid anion and one tertiary ammonium cation, plus the final hydrolyzing step to remove any residual anhydride groups which are not biocompatible to yield microparticles crosslinked with disulfide bonds, that are biocompatible and due to their polyampholyte character have desirable cryoprotective properties.

[0076]

[0073] In some embodiments, DMAPA can be replaced with other molecules comprising one primary amine group and one tertiary amine group, such as N,N-dimethylaminoethylamine, N,N- dimethylaminobutylamine, or N,N-diethylethylenediamine. In some embodiments, it may be desirable to introduce two or more cationic charges with every amine reacting with an anhydride moiety, and in such cases the amine would contain one primary amine and two or more tertiary amines, such as (2-N,N-dimethylaminomethyl)-4-amino-1-N,N-dimethylaminobutane (structure shown below), and other branched or linear tri or higher amines.

[0077]

[0074] In some embodiments, the amines used may comprise more than one primary amine. In these cases, some crosslinking may be expected but this may be mitigated by using these modifying amines in significant excess over the anhydride groups present on the microgels. Examples include tetraethylenepentamine, and other linear and branched oligoamines.

[0078]

[0075] In some embodiments, the disulfide bonds from both cystamine and optionally dimethacrylic crosslinkers (1) and / or (2), form at least 90, at least 95, at least 97, at least 98, or at least 99 % of the crosslinks within the microparticle formed. In some embodiments, the total percentage of actual disulfide crosslinks relative to total monomer units is in the range of 0.1 to 20 mol%, preferably 0.2 to 10 mol%, and most preferably 0.5 to 5 mol%.

[0079]

[0076] Swollen and suspended microgels having properties described herein may be directly combined with mammalian and other cells in culture, suitably, in a 10,000:1 to 1 :1 , and preferably 5000:1 to 2:1 , and most preferably a 500:1 to 5:1 volume ratio of microgels to cells. In some embodiments, the microgels form jammed gels upon swelling in media at microgel loadings of 1 to 20 wt / v %, and preferably 4 to 10 wt / v %. Jammed gels are close-packed arrays of soft particles that are solid-like or gel-like under low stress, but can flow under higher stress. Entrapment or immobilization of cells within the jammed gel can mitigate cryodamage to cells by reducing ice crystallization around the cells, and by partial dehydration of the cells during freezing and thawing. In another embodiment concentrated suspensions of highly swollen microgels form viscous solutions that prevent cell sedimentation and aggregation. In some embodiments, the microgels used for cryopreservation and 3D printing have an anionic:cationic ratio of between 80:20 - 20:80. In some embodiments, the hydrogel microparticles are in a concentration of 1 - 20 wt / v %, and preferably 4 to 10 wt / v %. This value can depend on the microgel stiffness and the solutions’ viscosity. Stiffer microgels capable of co-sedimenting with cells to form a jammed gel can be used effectively in the range of 5 - 20 wt / v % and softer microgels capable of forming a volume filling viscous solution that prevents cell sedimentation at a concentration range of 1 - 10 wt / v% that can be separated from cells by centrifugation. Effects of composition on microgel stiffness after dilution with media, is described in greater details in Sinjari, S., Freitag, J. S., Herold, C., Otto, O., Smith, D. M., & Stover, H. D. (2020). Tunable polymer microgel particles and their study using microscopy and real-time deformability cytometry. Journal of Polymer Science, 58(17), 2317- 2326 which is incorporated herein by reference in its entirety.

[0080]

[0077] During freezing and thawing, these microgels surround the cells, and prevent ice crystals from causing cell damage by penetrating cell walls. At the same time, slow freezing of the continuous cryo-media will lead to increased osmotic pressure in the microgels, which in turn will lead to partial dehydration of the cytosol of the embedded cells and cell clusters. This partial cell dehydration will likely start right upon incorporating the cells into the jammed gel, as the microgels in a jammed gel are by definition not yet swollen to equilibrium, and will hence tend to absorb additional water from their environment, including the cytosol of embedded cells. The resulting higher osmotic strength (higher protein concentration) within the cytosol will reduce ice crystal formation within the cells and cell clusters. Additionally, the microgels can reduce ice recrystallization during thawing, that could produce larger, cell-damaging ice crystals. In another embodiment, the addition of microgels under rapid freezing conditions leads to the vitrification of the continuous media inhibiting ice crystal formation.

[0081]

[0078] These microgels, by virtue of their relatively large size (1 - 10 micrometres) and nonfouling polyampholyte nature, are not likely to be taken up into mammalian cells by affinity- mediated processes or pinocytosis, and hence overcome key concerns with current cell cryoprotective agents such as dimethyl sulfoxide (DMSO) (used with cells including stem cells), and ethyleneglycol / glycerin and other sugar-derived molecules (used with cryostored blood), that is, residual cytotoxicity and the effect of the cryoprotective agents on the cell’s ability to differentiate (sternness) as well as time needed to remove intracellular cryoprotective agents.

[0082]

[0079] Mono-disperse microgel particles as described here enable better control over degree of deformation and hence help to minimize interstitial free aqueous phase in proximity to cells, and the associated risk of formation of cell-damaging ice crystals. Avoiding the formation of external ice crystals is important as these ice crystals can pierce cells membranes. Furthermore, the microgel of the present disclosure can also dehydrate the cytosol before and during freezing, thereby mitigating cell damage by ice crystal formation within cells.

[0083]

[0080] In some embodiments the soft, deformable polyampholyte microgels of the present disclosure can be used to fully or at least partially replace conventional cryo- protective agents that are cell-penetrating such as DMSO. This is particularly advantageous for cells that are sensitive to cryo-protecting cell penetrative agents (e.g., DMSO).

[0084]

[0081] As a result of the cleavage of the anhydride crosslinker, the microgels are highly swellable and deformable, with overall moduli approaching those of mammalian cells and tissues. These particles suitably may have no or very minimal extraneous surface residues of any stabilizer. These microgels have shown utility as non-cell penetrating cell cryoprotecting agents, as well as suspension agents helpful for storage and shipping, for both individual cells and cell clusters.

[0085]

[0082] In addition the polyampholyte microgels of the present disclosure can prevent rapid cell sedimentation to ensure cell survival during freeze-thaw processes encountered during cryostorage, as well as during accidental thaw events.

[0086]

[0083] These cell-sized hydrogel particles are much less likely to be taken up, by passive or active transport processes, by the cells than would be linear polymers of similar composition, reducing concerns such as cytotoxicity or interference with cellular differentiation.

[0087]

[0084] Microparticles as described herein may be further modified to improve their cryoprotective properties by three methods:

[0088]

[0085] Modifications with peptides can facilitate cell-microgel interactions and support cell functions by binding to integrin receptors. Crosstalk among different signaling pathways can act synergistically to enhance cellular responses such as cell adhesion, migration, or differentiation. Peptides derived from collagen (e.g. GFPGER (SEQ ID No:1), LAGSCLARTSTM (SEQ ID No:2)), fibronectin (PHSRN (SEQ ID No:3), GRGDSP (SEQ ID No:4), laminin (e.g. IKVAV (SEQ ID No:5), TWYKIAFQRNRK (SEQ ID No:6)), other RGD motifs such as SEQ ID No:7, Ac-G-R-G-D-I-P-A- S-S-K-G-G-G-G-S-R-L-L-L-L-L-L-R-NH2 (Peptide2000TM) and other extracellular matrix molecules (e.g. the fibrin-binding peptide NQEQVSP) can be synthesized and conjugated to the microgels.

[0086] The present microgels are also useful in 3D printing, particularly in the 3D printing of viscous, cell-containing solutions to form tissue-like cell-hydrogel combinations with a wide range of geometries. Specific implementations include printing self-supporting structures into a supporting matrix. These matrices are usually shear-thinning (ideally acting as a Bingham plastic), compatible with mammalian cells, and may allow curing of the printed hydrogel device prior to retrieval and further use. In some embodiments, the printed material, for example a patch, can be printed and frozen to -80°C using standard slow cooling techniques. When printing with cells, the microgel jammed gel can thus serve as both a shear-thinning support matric, and as a cryoprotective matrix for the formed 3D printed hydrogel comprising cells, helping to preserve the cell payload as well as the hydrogel, during freezing and thawing stages.

[0089]

[0087] A number of shear-thinning supporting baths for 3D printing are known in the art. These include self-associating polymers such as the family of Pluronics and other polymers, as well as assemblies of spheres commonly made of gelatin, known commercially as LifeSupport™ particles (these generally contain Type A gelatin that is dissolved with 11 mM CaCh, stirred and cooled into -spherical, 30 to 60 micrometer diameter, gelatin microparticles) that are used in Freeform Reversible Embedding of Suspended Hydrogels (FRESH) bioprinting. The latter also operates on the principle of jammed gels that become fluidized by the movement of the printing needle and thus offer little resistance to the placement of the bio-ink being printed, but subsequently revert to a jammed gel and thus minimize further movement and distortion of the printed hydrogel, acting as a Bingham plastic.

[0090]

[0088] One of the challenges and limitations in such 3D bioprinting is the impact of the LifeSupport™ spheres on the surface structure of the printed material. LifeSupport™ beads typically have diameters between 30 and 60 pm, and while they are deformable, they may result in large and unpredictable imprints into the surface of printed hydrogels.

[0091]

[0089] Another challenge encountered in printing into LifeSupport™ beads is the need to subsequently warm the printing bath to about 37°C in order to melt the LifeSupport™ beads, composed of gelatin, above their melting point. While this process is capable of breaking down the gelatin beads, it also can lead to incorporation of some of the gelatin into the printed hydrogel. This is not desirable for bioprinted hydrogels with defined compositions and clinical application.

[0090] A further challenge is that the design space of gelatin-based beads is limited by their need to maintain specific thermal characteristics, including a melting point not exceeding physiological temperatures.

[0092]

[0091] The present disclosure describes a preparation and composition of degradable microgel spheres that can overcome the above challenges and limitations. Specifically, they are formed by precipitation polymerization of methacrylic anhydride, optionally with a degradable disulfide-containing divinyl crosslinker, in suitable solvent systems. The resulting microgel spheres are narrow disperse in size, with diameters tunable to between approximately 1 and 10 micrometres. They are post-modified by reaction with cysteamine, a diamino-disulfide that can react with two anhydride moieties on different polymer chains to form de novo or additional crosslinks incorporating a disulfide unit. They are optionally subsequently post-modified by reaction with diamines comprising, at least one primary and at least one tertiary amine, such as 3-amino-1-dimethylaminopropane or similar di and oligo amines, giving them polyampholyte characteristics that render their swelling behaviour less sensitive to ions including calcium, strontium and barium ions. This post-modification simultaneously degrades the methacrylic anhydride crosslinks but leaves any disulfide crosslinks derived from disulfide-containing divinyl crosslinkers intact. The insensitivity of these polyampholyte microparticles to changes in ionic strength helps resist changes in the properties of the jammed gelling bath during printing in presence of 5 to 50 mM, and preferably 10 to 25 mM calcium chloride in the printing bath, and during post-print treatment of the printed patch by exposure to higher concentrations of calcium chloride, strontium chloride or barium chloride. Although polyampholytes are preferred to obtain the resistance, this is not necessary as sufficient resistance to 10 - 20 mM calcium chloride can be achieved with non-stoichiometric particles (e.g. 70% anionic and 30% cationic).

[0093]

[0092] The resulting disulfide crosslinked microgel particles are stable under normal physiological conditions for periods of at least one week, but are degraded rapidly upon addition of reducing agents including TCEP, THPP and glutathione at cell-compatible levels of 0.1 to 40, preferably 0.2 to 20, and most preferably 0.5 to 5 millimolar concentrations, for up to 60 minutes.

[0094]

[0093] There is also provided a method for printing gel formers comprising synthetic gel formers together with sodium alginate and optionally single cells or cell clusters such as islets of Langerhans, through a nozzle guided by a 3D printer system where the nozzle is immersed in a jammed gel bed of swollen microgels as described above. The printing is performed by a controlled movement of the nozzle tip and in some cases support bath in 3 dimensions, while extruding the gel formers, optionally containing cells, in order to generate regular 2D and, in particular, 3D patterns. Specifically, printing a lattice of evenly spaced linear string segments, with a vertical offset or increment identically to or close to the diameter of the string sections, can then lead to formation of a grid or patch with partially fused intersections of string sections in adjacent layers. The presence of crosslinkable groups on the gel formers can thus be used to covalently interconnect the patch segments at these junctions, and impart long-term resilience to the overall patch.

[0095]

[0094] The jammed gel made of disulfide-crosslinked polyampholyte microparticles acts as a shear-thinning printing bed to support the extruded string sections and in fact enable formation of a 3D printed patch. After printing is complete, the jammed gel supporting bath can be removed by a combination of washing and reductive degradation. Simple washing under gentle agitation can be used to remove most or all of the microgels. Any microgels remaining after washing may be removed through partial degradation during exposure to 0.1 - 5 mM TCEP for 2 to 30 minutes (or alternatively glutathione).

[0096]

[0095] The microgels used as printing bath may also be designed to partly shrink during exposure to higher levels of calcium, strontium or barium, such as the exposure to 100 mM calcium chloride used to stiffen the printed hydrogel during removal from the printing bath. Such calcium responsive microgels may be achieved through off-stoichiometric compositions, such as being comprised of a 10 / 90 to 40 / 60 ratio of cationic to anionic groups.

[0097]

[0096] Another advantage of using these disulfide-crosslinked microparticles as printing baths is that degradation with TCEP is a process already used in order to crosslink certain thiol - ene polymers through reductive deprotection of disulfide, including SPy and cystamine-protected thiols. Finally, disulfide crosslinked polyampholyte microgels are expected to also slowly degrade in vivo, through reductive action of naturally present compounds such as glutathione. This feature insures against any residual microgels that inadvertently remain with the printed hydrogels after implant.

[0098]

[0097] In one example, a printing of self-crosslinking hydrogel structures in jammed gel made of degradable microgels as presently described is performed. The printing can optionally be performed with coaxial needles, to form string sections having distinct, coaxial hydrogel cores and shells, with relative dimensions dependent on flow rates for extrusion of inner and outer layer. After the printing is complete, mechanical washing can be used to remove most of the microgels which can be followed by a degradation using TCEP in a concentration of 0.1 - 100 mM, 0.2 - 20 mM or 0.5 - 5 mM. In some cases, the TCEP treatment can be done at a concentration of 1 mM for around 20 minutes. The degradation or reducing treatment, which can be done with TCEP as described above, or THPP, or glutathione, is performed during the curing step after the microgel was printed (i.e. when it is allowed to cure). The patch is then transferred to a new gelling solution containing a gelling salt (e.g. barium, strontium or calcium chloride), and optionally excess cysteine to convert residual vinyl groups. Optionally, the patch can be cured fully by adding the gelling solution (calcium, strontium, zinc, iron, or barium salts) directly to the printing bath once the patch has been printed, and before removing the patch.

[0099]

[0098] Due to their small size (1 - 10 micrometer) and tunable deformability, the microgels described herein are improvements over currently utilized microgels such as LifeSupport™ FRESH particle arrays, particular with respect to enabling the formation of 3D printed hydrogels with smooth surfaces.

[0100]

[0099] The shape and size of the resulting printed material can vary. For example, a 2 x 2 cm, 3 x 3 cm, 4 x 4 cm, 5 x 5 cm, 6 x 6 cm or 7 x 7 cm patch can be printed which may be made of 1 to 10 layers. The concentration of the calcium chloride in the bath should be tuned depending on the printing shape and composition. For example, calcium chloride can be present in the printing bath in a concentration of from 3 to 100 mM, 5 to 50 mM, or 10 to 30 mM. Calcium can be replaced by strontium or barium. A strontium salt can be used in concentrations of 1.5 to 50 mM, 2.5 to 25 mM or 5 to 15 mM, and a barium salt can be used in concentrations of 1 to 35 mM, 2 to 20 mM, or 3 to 15 mM.

[0101]

[0100] One preferred configuration of a patch is to print square or rectangular shapes with non-equal side lengths, such as 1 by 2 cm or 4 by 6 cm. These shapes generally offer a better fit for the patient, and the corners could be filleted to limit the amount of sharp angles. Other shapes and configurations can be contemplated and the shape is not limitative.

[0102]

[0101] The printing bath is optionally supplemented with viscosity enhancers such as carboxypropyl cellulose, carboxymethyl cellulose, hydroxyethyl methacrylate, polyethylene glycol, pluronics, and, in particular, hyaluronic acid, for example from 0.2 to 10 wt. %, from 0.3 to 5 wt. %, or from 0.5 to 3 wt. %. The addition of hyaluronic acid has the advantage of decreasing the weight percent of microgels needed and has synergistic interaction with the formation of the microgel jammed gel. For example, addition of 1 wt.% hyaluronic acid allows formation of jammed gels suitable for 3D bioprinting at 5 wt. % microgels, as opposed to 10 wt.% in absence of hyaluronic acid.

[0103]

[0102] Yet another advantage is that the linear or branched polymers generated from the disulfide crosslinked microgels can attach to residual vinyl groups present on or within the outer layers of the linear string sections as well as the junctions, generating a polyampholyte antifouling layer on the outer surface of the hydrogel patch, while at the same time contributing to further crosslinking of the hydrogels, and capping of residual vinyl groups that were not used for initial crosslinking of the thiol / ene network. Similarly, high molecular weight linear or branched polyampholytes carrying disulfide groups may be infused into the printing bath, and thus be incorporated into the surface of the curing patch during reductive treatment with, e.g., TCEP, THPP and glutathione. These options represent significant advances over gelatin-based jammed gels, where thermal melting of the printing support bath results in unavoidable impregnation of the 3D printed hydrogel with gelatin.

[0104]

[0103] Additional functionalization can optionally be performed with diol or diamine compound crosslinkers. Examples of such compounds are provided below with “x” and “y” denoting repeating units. In addition, acrylates and methacrylates, as well as acrylamide and methacrylamides derivatives of such compounds, can serve as di or oligo vinyl crosslinkers that can be added during the initial particle formation. Compounds A-J have only a portion of the hydroxy or amine groups being modified with vinyl ((meth)acrylate or (meth)acrylamide) groups. These compounds can be added during initial particle formation to be incorporated by mechanisms involving both attack of the hydroxy or amine group on anhydride, and incorporation of the vinyl group by radical copolymerization. Specifically, although compounds A-J are not monomers they can react in situ with methacrylic anhydride to form their respective bis (meth)acryloyl or bis or tetra(meth)acrylamide derivatives as monomers. Alternatively, they can be previously reacted with methacrylic anhydride or methacryloylchloride prior to precipitation polymerization to form their bis (meth)acryloyl or bis or tetra(meth)acrylamide derivatives as monomers.

[0104] Wherein x and y are each independently an integer from 0 to 7, from 1 to 7, from 0 to 5 or from 1 to 5. As explained above, formulas 1 , 2, and A-J can be used at the start of the process during the particle formation in order to directly obtain a microparticle having the disulfide bonds. The particle formation is performed under heating with a marginal solvent as described above. Accordingly, a polymerization can be performed with methacrylic anhydride and also with the formulas 1 , 2 or A-J incorporating disulfide bridges. In such embodiments, the second step of functionalization with cystamine becomes optional because some disulfide crosslinks are already incorporated during polymerization, but can be beneficial as it allows greater control over placement of the disulfide crosslinker within the microgel. The remaining steps are as explained above of reacting the microparticle with a compound comprising at least one primary amine group and at least one tertiary amine group, and hydrolyzing any remaining anhydride groups on the microparticle by contacting the microparticle with an alkaline aqueous solution to obtain the microgel particle.

[0105]

[0105] A further example is that the microgel jammed gels are optically transparent, which allows for direct observation of the string segments as they are formed. This allows rapid in-situ determination of attributes such as string diameter, over extrusion, string slippage, deformations, cell centering, and other features.

[0106]

[0106] One exemplary in vivo application for the microgels is as vaccine carriers. In some embodiments, the microgels associate or encapsulate a vaccine composition and can therefore serve as delivery platform for antigens. Accordingly, the microgels may:

[0107] • Act as carriers for antigens, e.g., RNA including m-RNA, DNA, proteins, viral shell fragments, whole inactivated viral shells, or active innocuous viruses such as adenoviruses that have been modified to express the desired antigen protein;

[0108] • Have cationic groups that can electrostatically bind the antigen during storage and administration to the recipient’s immune system;

[0109] • Have adjuvant properties to ensure recognition and processing by the host immune system, including where said adjuvant properties are based on cationic or polycationic groups, or on certain carbohydrate groups; • Have compositions where cationic or polycationic groups can be cleaved by spontaneous or enzyme-mediated hydrolysis in order to release the bound antigen payload over a timeframe beneficial to evoking a strong immune response in the recipient;

[0110] • Have crosslinkers that may undergo slow spontaneous or enzyme-mediated hydrolysis in order to ensure ultimate clearance of the microparticles from the recipient through processes including renal clearance;

[0111] • Have compositions including cationic and polycations groups as well as non-stoichiometric polyampholytes that can bind antigen for storage at room temperature defined as up to 40 °C, and without need for cold-chain logistics during storage and transportation; and

[0112] • May additionally be loaded with silver nanoparticles to enhance cellular immune response, either by coprecipitation during precipitation polymerization, or by reductive precipitation from silver salts as part of the post-functionalization, or by adsorption of preformed silver nanoparticles onto the described polymer nanoparticles.

[0113]

[0107] Accordingly, in some embodiments, there is provided a vaccine composition or a therapeutic composition comprising the microgels of the present disclosure. The microgels of the present disclosure can be administered to a subject in need thereof without a further carrier. For example, the polycation carrier particles can be suspended in a solution for intramuscular, intranasal, intracutaneous and percutaneous administration. In other embodiments, the microgel particles may be formulated as part of an oral composition (e.g. an oral capsule) or for nasal application, for transport across the relevant mucosal membranes.

[0114]

[0108] In some embodiments, the microgels are complexed with, or have attached, or encapsulate, a cargo or payload that elicits a specific immune response, and are administered to a subject. The cargo can be a nucleic acid, such as DNA and RNA or a protein or proteinaceous molecule or material. Accordingly, in other embodiments, there is provided a method of delivering antigens to cells by administering the microgels of the present disclosure having a payload.

[0115] EXAMPLE 1

[0116]

[0109] In a 1 L round-bottom flask, 840 mL of a mixture of MEK / Heptane (57.5142.5 vol% = 483 mL / 357 mL respectively) was prepared. Then 35 g (0.227 mol, 4 w / v%) of methacrylic anhydride as sole monomer and 2.11 g (8.51x10-3mol, 3.75 mol% relative to methacrylic anhydride) of 2,2’-azobis(2,4-dimethylvaleronitrile) (ADVN, initiator) were weighed in to separate glass vials. 30 mL of the MEK / Heptane mixture was poured into the vial that contained the ADVN for dissolution. The monomer was combined with the remaining solvent mixture in a 1 L glass reactor pre-heated to 50 °C using a water heating bath. The temperature of the reactor was stabilized over a period of 1 hour under nitrogen bubbling and stirred with a large overhead 3- bladed marine propeller stirrer at 20 rpm. After the temperature stabilized, the nitrogen bubbling was stopped, and the pre-dissolved initiator was added. Within about 100 minutes the reaction mixture turned milky due to formation of particles, and the polymerization process was continued for 16 h. The particle suspension was transferred to 400 mL centrifuge containers and centrifuged for 15 min at 3080 G to spin down the polymer particles. The supernatant was discarded, and the particles were resuspended three times in acetone and centrifuged for 15 min at 3080 G. Imaging of the particles by optical microscopy showed spherical particles with about 1 micrometer diameter (Figs. 1A-1 B). A 1 mL aliquot of the particles dispersed in acetone was taken and evaporated to dryness to estimate the obtained particle mass. The microparticles had a structure as shown below, with “x” and “y” and “z” independently denominating repeating units. For simplicity, each unit is shown once however it should be understood that the different units illustrated can be in any order and intertwined.

[0117]

[0110] For the post-modification step, 7.3 g (4.86 x 10'2mol) of the microgels were dispersed in 300 mL of N,N-dimethylformamide (DMF) in a 1 L round bottom flask. The dispersion was stirred using a magnetic stir bar and an excess of triethylamine (4.92 g, 0.048 mol) was added. In a 20 mL glass vial, 0.767 g (3.40 x 10'3mol) of cystamine dihydrochloride (7 mol%, crosslinker) was weighed out and dissolved in 5 mL of dimethylsulfoxide (DMSO), added to the reaction mixture and stirred for 5 hours. After 5 hours, an excess of 3-(dimethylamino)-1-propylamine (DMAPA, 14.91 g, 0.14 mol) was added and the reaction was stirred overnight. The reaction mixture was transferred to 400 mL containers and centrifuged for 10 min at 3080 G to sediment the particles. The sedimented particles were then resuspended three times in DMF and centrifuged for 10 minutes to remove unreacted amines. The particles were transferred to water to hydrolyze residual anhydride groups, and after complete swelling, the mixture was transferred to cellulose dialysis bags with 12-14 kDa molecular weight cut-off. The dialysis was carried out for two days against saline and then for two more days against distilled water. After dialysis was completed the pH of the microgel suspension was adjusted to 7 using 1 M HCI, and transferred into 50 mL centrifuge tubes, frozen using dry ice for 2 hours and freeze-dried for 4 days. The dried samples were weighed, and yields were calculated to be about 50% for the total process). Imaging of the microgels showed spherical particles with a diameter of 2 micrometres.

[0118] Scheme of the post modification steps to form cystamine-crosslinked polyampholyte microgels

[0119]

[0111] A portion of the microgel particles were fluorescently labelled as follows: fluorescein cadaverine dihydrochloride (0.025 mol%) was pre-dissolved in DMF in the presence of tri ethyl amine, and immediately added to the reaction mixture and reacted for 30 min before introducing the excess of DMAPA. The confocal images are presented in Figs. 2A-2B which show fluorescently labelled particles, imaged in aqueous suspension at pH 7 after reaction with cysteamine and DMAPA, and following hydrolysis of residual anhydride groups.

[0120]

[0112] Sterilization of samples is necessary for experiments with biological material. Following common procedure, freeze-dried microgels in centrifuge tubes were resuspended by addition of 45 mL of 70% ethanol. The suspension was left 2 hours, then centrifuged for 5 minutes at 4000 RPM to sediment the sterile microgels. The tubes were transferred into a biosafety cabinet for sterile work. The 70% ethanol supernatant was removed and the microgels were reswollen by adding sterilized water into the centrifuge tubes. The centrifuge tubes containing the sterile aqueous microgel suspension was closed with sterile freeze-drying closures, frozen in dry ice for 2 hours, transferred into freeze-drying flasks, and freeze-dried for 4 days.

[0121] EXAMPLE 2

[0122]

[0113] In the present example, the initial particles are formed by precipitation copolymerization of methacrylic anhydride with a dimethacryloyl disulfide crosslinker. The benefit of this combination crosslinking is that it provides two separate means of introducing crosslinker into the microgel particles. In particular, under circumstances where post-modification with cystamine seems to favour crosslinking at the particle surface, combination of both types of crosslinking allows better control of placement of crosslinking groups throughout the particles.

[0123]

[0114] In a 500 mL media bottle, 500 mL of a mixture of MEK / Heptane (57.5% / 42.5% = 287 mL / 212 mL respectively) was prepared. Then 20 g (0.12 mol) of methacrylic anhydride (4% w / v), 1.13 g of dimethacryoyl disulfide (3 mol%, 1.13 x 10'3mol), and 1.21 g of 2,2’-azobis(2,4- dimethylvaleronitrile) (ADVN, 3.75 mol% relative to monomer, 4.86 x 10'3mol) were weighed into three different glass vials. 18 mL of the MEK / Heptane mixture was taken and poured in the vial that contained the ADVN for dissolution. The monomer, the crosslinker and the pre-dissolved initiator were added to the remaining solvent mixture contained in the 500 mL glass media bottle. The glass media bottle was placed into a hybridizer stabilized at 53 °C and gently rolled using a small hotdog roller for mixing, between 90 to 105 min passed to see the formation of particles in the reactor, the polymerization process went for 16 h.

[0115] The hybridizer was turned off and the reactor was cooled to room temperature, then the liquid mixture containing the white particles were transferred to 400 mL containers and centrifuged for 15 min at 3080 G to spin down the polymers, the supernatant was discarded. The particles were washed with acetone to remove any unreacted monomer and centrifuged for 15 min at 3080 G three times. Imaging of the particles was done showing spherical morphology and 1 micrometer diameter (Figs. 3A-3B). An aliquot of 1 mL of the particles dispersed in acetone was dried to estimate the obtained mass.

[0124]

[0116] In a post-modification step, 15.00 g (0.10 mol) of the microgels were dispersed in 700 mL of DMF and poured in a 1 L round bottom flask, the dispersed microgels were stirred using a magnetic stir bar and triethylamine (10.11 g ,0.1 mol) was added in excess. In a 20 mL glass vial, 1.12 g (5 x 10'3mol) of cystamine dihydrochloride (5 mol%, crosslinker) was weighed and dissolved in 20 mL of DMF in the presence of an equimolar quantity of DMAPA (0.50 g, 4.97 x 10'3mol) by stirring for 10 min, added to the reaction mixture and stirred for 5 hours. After the 5 hours, an excess of 3-(dimethylamino)-1-propylamine (DMAPA) (30.65g, 0.30 mol) was added and the reaction was stirred overnight. The reaction mixture was transferred into 400 mL containers and centrifuged for 10 min at 3080 G to sediment the polyampholyte particles, then washed with DMF and centrifuged for 10 minutes three times to remove unreacted amines. Water was added to the microgels to hydrolyze residual anhydride groups, and after complete swelling of the microgels the sample was transferred into a dialysis bag with 12-14 kDa molecular weight cut off. The dialysis process was carried out for four days, two days in saline and two days in distilled water. After dialysis the pH of the microgel solution was adjusted to 7 with 1 M HCI and transferred into 50 mL centrifuge tubes, frozen using dry ice for 2 hours and freeze-dried for 4 days. The dried samples were weighed, and yields were calculated (usually over 50% yield for the total process). Imaging of the microgels showed spherical morphology and a diameter of 2 micrometres (Figs. 4A-4B).

[0125] Scheme of the modification step for the synthesis of polyampholyte microgels using both dimethacryloyl disulfide and cystamine:

[0126]

[0127]

[0117] The microgels were fluorescently labelled as follows: fluorescein cadaverine dihydrochloride (0.025 mol%) was pre-dissolved in DMF in the presence of triethylamine, and immediately added to the reaction mixture and reacted for 30 min before introducing the excess

[0128] DMAPA.

[0129]

[0118] Sterilization of samples is performed before cryogenic use. In a common procedure, 45 mL of a 70% ethanol solution is poured into centrifuged tubes that contain dried polyampholyte microgels for 2 hours, they are then centrifuged for 5 minutes at 4000 g to sediment the microgels. In a BSC, the 70% ethanol solution is removed from the sample and the microgels are reswollen in sterilized water. After reswelling, the samples were frozen with dry ice for 2 hours, transferred into freeze-drying flask in the BSC and dried for 4 days.

[0130] EXAMPLE 3

[0131]

[0119] This example describes a microgel that is crosslinked using crosslinker (1), the ranges of crosslinker explored were between 3-7 mol%, resulting MG were modified with UPy. The range of UPy was varied between 0.1- 20 mol% and preferably between 1 to 10 mol%.

[0132]

[0120] In a post-modification step, 1.0 g (6.67x10-3mol) of the microgels were dispersed in 50 mL of DMF in a glass vial, microgels were stirred using a magnetic stir bar and triethylamine was added in excess (0.74 g, 7.33x10-3mol). In a 20 mL glass vial, 0.089 g (3.33x10-4mol) of 1- (6-aminohexyl)-3-(6-methyl-4-oxo-1,4-dihydropyrimidin-2-yl)urea (UPy) was weighed and dispersed in 5 mL of DMF by heating for 30 minutes at 85 °C, and then added to the reaction mixture and stirred overnight at room temperature.

[0133]

[0121] For this example, DMAPA was not added in order to explore the effect of UPy on the overall properties of the microgel. The reaction mixture was poured into 50 mL centrifuge tubes and centrifuged for 10 min at 3080 G to sediment the microparticles, then resuspended in acetone and centrifuged for 10 minutes three times. Water was added to the microgels to hydrolyze them and the mixture was stirred overnight at 85 °C, after complete swelling of the microgels the sample was transferred into a dialysis bag with 12-14 kDa molecular weight cut off. Microgels were dialysed for four days, two days in the presence of NaCI and two days in distilled water. After dialysis the pH of the microgel solution was adjusted to 7 using a 1 M solution of HCI and transferred into 50 mL centrifuge tubes, frozen using dry ice for 2 hours and freeze-dried for 4 days. The dried samples were weighed, and yields were calculated (usually over 50% yield for the total process). Imaging of the microgels showed spherical morphology and a diameter of 3.5 pm (Fig. 5).

[0134] Scheme of the functionalization of microparticles using UPy

[0135] EXAMPLE 4

[0136]

[0122] This example describes a microgel that is crosslinked using crosslinker (1), the ranges of crosslinker explored were between 3-7 mol%, and the resulting microgels were modified with dopamine. The amount of dopamine ranges from 0.1- 25 mol% and preferably between 1 and 10 mol%.

[0137]

[0123] In a post-modification step, 1.0 g (6.67x1 O'3mol anhydride) of each microgel type was dispersed in 50 mL of DMF in a glass vial, microgels were stirred using a magnetic stir bar and a slight excess of triethylamine (0.74 g, 7.33x1 O'3mol) relative to anhydride was added. In a 20 mL glass vial, 0.316 g (1.66x1 O'3mol, 25 mol% relative to anhydride) of dopamine was weighed and dissolved in 5 mL of DMF, and then added to the microgel suspension. After 5 hours, an excess of 3-(dimethylamino)-1-propylamine (DMAPA) (2.04 g, 0.02 mol) was added and the reaction was stirred overnight. The reaction mixture was poured into a 50 mL centrifuge tube and centrifuged for 10 min at 3080 G to sediment the microparticles, then resuspended in DMF and centrifuged for 10 minutes three times to remove unreacted amines. Water was added to the microgels to hydrolyze them and after complete swelling of the microgels the sample was transferred into dialysis bag with 12-14 kDa molecular weight cut off. Microgels were dialysed for four days, two days in the presence of NaCI and two days in distilled water. After dialysis the pH of the microgel solution was adjusted to 7 using a 1 M solution of HCI and transferred into 50 mL centrifuge tubes, frozen using dry ice for 2 hours and then freeze-dried for 4 days. The dried samples were weighed, and yields were calculated (usually over 50% yield for the total process). Imaging of the microgels showed spherical morphology and a diameter of 3.0 pm (Fig. 6).

[0138] Scheme of the functionalization of microparticles using dopamine and DMAPA

[0139] EXAMPLE 5

[0140]

[0124] This example provides a microgel that is crosslinked using crosslinker (1), the ranges of crosslinker explored were between 3-7 mol%, resulting microgels were modified with peptides, i.e. RGD. The amount of peptide ranged from 0.1- 20 mol% and preferably between 1 and 10 mol%.

[0141]

[0125] In a post-modification step, 1.0 g (6.67x10-3mol) of the microgels were dispersed in 50 mL of DMF in a glass vial, microgels were stirred using a magnetic stir bar and triethylamine was added in excess (0.74g, 7.33x10-3mol). In a 20 mL glass vial, 1.61 mg (4.666x10-6mol) of RGD was weighed and dispersed in 5 mL of DMF, and then added to the reaction mixture and stirred overnight at room temperature. For this example, DMAPA was not added, in order to be able to explore the contribution of just RGD alone to the properties of the microgel. The reaction mixture was poured into 50 mL centrifuge tubes and centrifuged for 10 min at 3080 G to sediment the microparticles, then washed with acetone and centrifuged for 10 minutes three times. Water was added to the microgels to hydrolyze them, after complete swelling of the microgels the sample was transferred into dialysis bag with 12-14 kDa molecular weight cut off. Microgels were dialysed for four days, two days in the presence of NaCI and two days in distilled water. After dialysis the pH of the microgel solution was adjusted to 7 using a 1 M solution of HCI and transferred into 50 mL centrifuge tubes, frozen using dry ice for 2 hours and freeze-dried for 4 days. The dried samples were weighed, and yields were calculated (usually over 50% yield for the total process). Imaging of the microgels showed spherical morphology and an average diameter of 3.9 pm (Fig. 7).

[0142] Scheme of the functionalization of microparticles using RGD

[0143] EXAMPLE 6

[0144]

[0126] All work related to the cryopreservation of cells was done in a sterile biosafety cabinet. Microgels were reconstituted at 2-10 wt% in sterile HypoThermosol™. The aqueous solutions contained 0-2% DMSO.

[0145]

[0127] Immortalized fibroblasts were frozen as single cells. Cells were carefully mixed with the reconstituted microgels at a concentration of 1 ,000,000 cells / mL and stored in 2mL Cryovials at a volume of 1000 pL. Cells were frozen using a Mr. Frosty™ at a rate of 1 °C / minute and stored at -80°C for up to 3 months. For longer storage times, liquid nitrogen is preferred.

[0146]

[0128] Cells in the cryovials were thawed quickly at 37°C, within 2-5 minutes. A small ice pellet remained before transferring to a sterile environment. The cell suspension was gently and slowly transferred into 10 X volume of medium, drop by drop. Cells were centrifuged at 300 x g for 5 minutes at room temperature (15 - 25°C). The supernatant above the microgel was carefully removed with a pipette. To degrade the microgels, 500 pL of 3 mM TCEP was added to 1000 pL of cells and microgel and incubated at 37°C for 5 minutes. Cells were centrifuged at 300 x g for 5 minutes at room temperature (15 - 25°C). The supernatant containing degraded microgels was carefully removed with a pipette, leaving a small amount of medium to ensure the cell pellet was not disturbed. One mL of culture medium containing 10 pM Y-27632 was added to the tube and mixed gently. Cells were plated onto tissue culture treated plates and flasks. The final cell concentration was between 3-5x103viable cells / cm2. If needed, microgel clusters were removed using a 40 pm cell strainer.

[0147]

[0129] As seen in Figs. 8A-8D, single fibroblasts show good viability after thawing when frozen in different microgel formulations with subsequent attachment and growth at rates comparable to commercially available solutions. Viability was preserved even with no (Fig. 8B) or low concentrations of DMSO (<2%) (Fig. 8C).

[0148] EXAMPLE 7

[0149]

[0130] Small induced pluripotent stem cell clusters of 10-100 cells were carefully mixed with the reconstituted microgels at a concentration of 1.000.000 cells / mL and stored in 2mL Cryovials at a volume of 1000pL. Cells were frozen using a Mr. Frosty at a rate of 1°C / minute and stored at -80°C for up to 3 months. For longer storage times, liquid nitrogen is preferred.

[0150]

[0131] Clusters in the cryovials were thawed quickly at 37°C, within 2-5 minutes. A small ice pellet remained before transferring the cryovial to the biosafety cabinet. The cell suspension was gently and slowly transferred into 10 X volume of medium, drop by drop. Cells were centrifuged at 300 x g for 5 minutes at room temperature (15 - 25°C). The supernatant above the microgel was carefully removed with a pipette. To degrade the microgels, 500 pL of 3 mM TCEP was added to 1000 pL of cells and microgel and incubated at 37°C for 5 minutes. Cells were centrifuged at 300 x g for 5 minutes at room temperature (15 - 25°C). The supernatant containing degraded microgels was carefully removed with a pipette, leaving a small amount of medium to ensure the cell pellet was not disturbed. One mL of culture medium containing 10 pM Y-27632 was added to the tube and mixed gently. Cells of one vial were plated onto one vitronectin coated 6-well plates.

[0151]

[0132] Figs. 9A-9D depict small iPSC clusters frozen with degradable microgels, showing increased viability compared to the negative and positive control. Increased attachment at day 1 was also observed, compared to controls. Negative control was hypothermosol, and positive control was CS10.

[0152] EXAMPLE 8

[0133] Single cell human mesenchymal stromal cells were carefully mixed with the reconstituted suspension of microgels that had been filtered through a 20 pm filter, at concentrations of 100,000-10,000,000 cells / mL and stored in 2 mL cryo-vials at a volume of 1000 pL. Cells were frozen using a Mr. Frosty at a rate of 1 °C / minute and stored at -80°C for up to 3 months.

[0153]

[0134] Frozen ells in the cryovials were thawed quickly at 37°C, within 2-5 minutes. A small ice pellet remained before transferring the cryovial to the biosafety cabinet. The cell suspension was gently and slowly transferred into 10 X volume of medium, drop by drop. Cells were centrifuged at 300 x g for 5 minutes at room temperature (15 - 25°C). The supernatant above the microgel was carefully removed with a pipette. The middle phase containing microgels was then carefully removed with a pipette, leaving a small amount of medium to ensure the cell pellet at the very bottom was not disturbed. One mL of culture medium containing 10 pM Y-27632 was added to the tube and mixed gently. Cells from one vial were plated onto one vitronectin coated 6-well plate.

[0154]

[0135] Figs. 10A-10D depict human mesenchymal stromal cells frozen with degradable microgel, showing comparable viability and attachment one day after thawing compared to the positive control while using less DMSO. Microgels without DMSO showed similar attachment and viability to the negative control Hypothermosol, but demonstrated increased proliferation over the next few days.

[0155] EXAMPLE 9

[0156]

[0136] IPSC cell clusters were carefully mixed with the reconstituted microgels at concentrations of 600,000-1 ,000,000 cells / mL and stored in 2 mL cryovials at a volume of 1000 pL. Cells were frozen using a Mr. Frosty at a rate of 1 °C / minute and stored at -80°C for up to 3 months. For longer storage times, liquid nitrogen is preferred.

[0157]

[0137] Cell clusters in the cryovials were thawed quickly at 37°C, within 2-5 minutes. A small ice pellet remained before transferring the cryovial to the biosafety cabinet. The cell suspension was gently and slowly transferred into 10 X volume of medium, drop by drop. The cell clusters were separated from the microgel by leaving the tube containing the cells and microgel with the medium standing until the cell clusters settled at the bottom, approximately 5 minutes. The supernatant above the microgel was carefully removed with a pipette, leaving a small amount of medium to ensure the cell / microgel pellet was not disturbed. To degrade the microgels, 500 pL of 3 mM TCEP was added to 1000 pL of cells and microgel and incubated at 37°C for 5 minutes. Cell clusters were allowed to settle for 5 minutes at room temperature (15 - 25°C). The supernatant containing degraded microgels was carefully removed with a pipette, leaving a small amount of medium to ensure the cell pellet was not disturbed. Thawed cell clusters were plated onto non-adhesive 6-well plates.

[0158]

[0138] Figs. 11A-11 B show human donor islets frozen in degradable microgel demonstrating similar viability to islets frozen in a commonly used freezing media.

[0159] EXAMPLE 10

[0160]

[0139] To create hybrid clusters 600,000 human induced pluripotent stem cells were reaggregated with Peptide2000TM-modified, non-degradable microgels at a concentration of 0.05 - 0.5 wt% using 24 well format Aggrewells™. Cells were incubated at 37°C for 6 days. The hybrid clusters were carefully washed out of the Aggrewells™ and plated onto non-adhesive wells for imaging.

[0161]

[0140] Fig. 12 shows examples of hybrid clusters of human induced pluripotent stem cells and Peptide2000™-modified microgel, demonstrating that the microgel is interacting with the cells and contributes to the formed cluster. Specifically, the figure shows a pictures with the xy projections demonstrating the microgel within the cell cluster.

[0162] EXAMPLE 11

[0163]

[0141] Cell clusters of 1000 to 3000 cells (150 - 300 pm) were mixed with the reconstituted microgels at approximately 200000 cells / mL. Cells were stored at room temperature and 4°C for three days to mimic transport. Cells were analyzed within the microgel mixture.

[0164]

[0142] For recovery, the cell clusters were gently and slowly transferred into 10 X volume of medium. The clusters were separated from the microgel by leaving the tube containing the cells and microgel with the medium standing until the clusters settled at the bottom, approximately 5 minutes. The supernatant above the microgel was carefully removed with a pipette. To degrade the microgels, 500 pL of 3 mM TCEP was added to 1000 pL of cells and microgel and incubated at 37°C for 5 minutes. Clusters were allowed to settle for 5 minutes at room temperature (15 - 25°C). The supernatant containing degraded microgels was carefully removed with a pipette, leaving a small amount of medium to ensure the cell pellet was not disturbed. Thawed clusters were plated onto non-adhesive plates.

[0143] Fig. 13 shows the human donor islets were suspended in microgels and transported for 3 days. Compared to islets transported in media, the microgel minimized aggregation and supported higher viability.

[0165] EXAMPLE 12

[0166] Synthetic procedure for the synthesis of PEG modified microgels.

[0167]

[0144] Amino-terminal PEG molecules in the range of 750 -10.000 kDa were used, including a low molecular weight (750 Da) oligomer and a high molecular weight polymer (10 kDa).

[0168] Synthetic procedure for the synthesis of PEG anionic microgels.

[0169]

[0145] In a round bottom flask, 2.74 g (0.0167 mol) of microgels were dispersed in 250 mL of DMF and stirred for 30 min; 1.86 g (0.0814 mol) of triethylamine were pre-weighed, dispersed in 3 mL of DMF and added dropwise to the reaction flask and stirred for another 30 mins. 0.73 g (10 mol%, 1.674x1 O'3mol) of amino terminated- PEG (750 Da or 10kDa) were weighed and dissolved in a mixture of ACN:DMF (1 :2) and added to the reaction dropwise. The reaction was stirred overnight. The sample was transferred into 50 mL conical tubes and centrifuged at 4000 rpm for 10 minutes. The crude product was washed another two times with 25 mL of DMF and centrifuged at 4000 rpm for 10 minutes. The precipitate was swollen in distilled water and stirred overnight. After this period, the sample was filtered twice using a 20 pm and a 10 pm membrane filter, and dialyzed for two days against saline and another two days against distilled water. The aqueous dispersion was then transferred into 700 mL containers and centrifuged for 2 hrs at 4000 rpm to separate the different components of the mixture. The upper layer contained the soluble mixture (linear and branched polymer), and the lower layer corresponds to the microgels. Both layers were separated, stored in conical tubes and freeze-dried.

[0170]

[0146] Samples modified with the 10 kDa molecular weight PEG contained some clusters that could be broken by stirring or vortexing. The sample modified with 750 Da molecular weight PEG did not contain any visible aggregates (Fig. 14).

[0171] Scheme. Reaction scheme for the synthesis of anionic microgels modified with PEG EXAMPLE 13

[0172]

[0147] The size, dispersity, and controlled degradability of the microgels, along with their viscosity in their jammed state in aqueous solutions at concentrations typically >5 wt. / vol.% (wt / v%), allows for their use as supporting baths for 3D bioprinting in cell-relevant conditions. Supporting baths are used to print materials or bioinks that are not strong enough immediately after extrusion to support the printed form. This printing methodology is particularly useful for hydrogels that form via crosslinking that at least partly occurs after printing, such as Ca2+- mediated crosslinking of alginate hydrogels, and hydrogels comprising polymers that crosslink after partial fusion at intersect points. Additionally, it is highly desirable that the supporting bath can be easily removed via a cell-friendly mechanism without impacting the structure of the printed hydrogel.

[0173]

[0148] This example provides microgels that are crosslinked using crosslinker (1), the ranges of crosslinker explored were between 3-7 mol%, and the resulting microgels were modified with DMAPA to generate amphiphilic microparticles.

[0174]

[0149] To assess the capacity of these jammed microgels as support baths for 3D printing, microgels were dissolved at 10 wt / v% in HEPES-buffered saline (HBS) with 22 mM Ca2+and used as a supporting bath for printing a solution of uncrosslinked 2 wt.% alginate, a structure that will only form a hydrogel in calcium solutions. The 2 wt.% alginate was extruded at 1.5 pL / s through a 20G nozzle with the tip moving at 9.5 mm / s using a Cellink BioX bioprinter. The patch printed was a 150 mm x 150 mm 3-layer patch with a 30% infill density. After printing, the patch was left to sit for 30 minutes to allow the entire patch to crosslink. Subsequently, the patch was rinsed with HBS and placed in a solution of 20 mM THPP for 30-60 minutes to dissolve any remaining microgels. The resulting patch was imaged and assessed in terms of the surface roughness of the filaments along with overall patch fidelity (Figs. 15A-15B).

[0175]

[0150] As shown in Figs. 15A-15B, which are images of a 2% alginate 150 mm x 150 mm 3- layer patch after printing in 10 wt / v% amphiphilic microgels crosslinked with (1), curing the alginate with 100 mM Ca2+, and subsequently degrading the hydrogels with 20 mM THPP, resulting in only the cured alginate patch. Fig. 15A shows 10X images display the roughness of these patches, focusing on all three layers of a given intersection indiviually on the right, which is significantly less than prints in a commonly used support bath system, LifeSupport™, owing to the smaller size of the microgels in comparison to the gelatine LifeSupport™ particles. Fig. 15B shows a stitched image of smaller 4X images of the whole patch which shows the integrity and consistency of the overall 2% alginate patch.

Claims

WHAT IS CLAIMED IS:1 . A method for producing a microgel particle, the method comprising: heating a marginal solvent comprising methacrylic anhydride monomers to polymerize the monomers and obtain a microparticle; crosslinking the microparticle with cystamine; reacting the microparticle with a compound comprising at least one primary amine group and at least one tertiary amine group, and hydrolyzing any remaining anhydride groups on the microparticle by contacting the microparticle with an alkaline aqueous solution to obtain the microgel particle.

2. The method of claim 1 , wherein the marginal solvent is selected from heptane, toluene, xylenes, methyl ethyl ketone (MEK), tetrahydrofuran (THF), acetonitrile, ethyl acetate, benzene, N,N-dimethylformamide (DMF), 1 ,4-dioxane, acetone, cyclohexane, or mixtures thereof.

3. The method of claim 1 or 2, wherein the heating is performed at a temperature of from 20 to 85 °C, preferably 40 to 65 °C, and most preferably 50 to 55 °C.

4. The method of any one of claims 1 to 3, wherein the marginal solvent further comprises a polymerization initiator, preferably activated by thermal stimuli, light or a redox process.

5. The method of any one of claims 1 to 4, wherein the steps of heating and crosslinking are performed concurrently.

6. The method of any one of claims 1 to 5, wherein the microparticle is crosslinked by disulfide bonds.

7. The method of any one of claims 1 to 6, further comprising between the steps of crosslinking and reacting, further reacting the microparticle with modifying compounds that increase the hydrogen bonding capacity.

8. The method of any one of claims 1 to 7, wherein the compound according has the formula NH2-R-N(R)2 where each R is independently C1-C6 alkyl that are each independently optionally substituted, optionally interrupted, and / or optionally terminated by S, O or N.

9. The method of any one of claims 1 to 8, wherein the compound is dimethylaminopropylamine (DMAPA), dimethylaminoethylamine (DMAEA) or dimethylaminobutylamine (DMABA).

10. A method for producing a microgel particle, the method comprising: heating a marginal solvent comprising methacrylic anhydride together with one or more of a crosslinker of formula (1) and / or (2), or a compound selected from compounds A, B, C, D, E, F, G, H, I, J, and combinations thereof, and a bis (meth)acryloyl or bis or tetra(meth)acrylamide derivative thereof, to obtain a microparticle;wherein x and y are each independently an integer from 0 to 7;reacting the microparticle with a compound comprising at least one primary amine group and at least one tertiary amine group, and hydrolyzing any remaining anhydride groups on the microparticle by contacting the microparticle with an alkaline aqueous solution to obtain the microgel particle.

11. The method of claim 10, wherein the marginal solvent is selected from heptane, toluene, xylenes, methyl ethyl ketone (MEK), tetrahydrofuran (THF), acetonitrile, ethyl acetate, benzene, N,N-dimethylformamide (DMF), 1 ,4-dioxane, acetone, cyclohexane, or mixtures thereof.

12. The method of claim 10 or 11 , wherein the heating is performed at a temperature of from 20 to 85 °C, preferably 40 to 65 °C, and most preferably 50 to 55 °C.

13. The method of any one of claims 10 to 12, wherein the marginal solvent further comprises a polymerization initiator, preferably activated by thermal stimuli, light or a redox process.

14. The method of any one of claims 10 to 13, further comprising between the steps of crosslinking and reacting, further reacting the microparticle with modifying compounds that increase the hydrogen bonding capacity.

15. The method of any one of claims 10 to 14, wherein the compound according has the formula NH2-R-N(R)2 where each R is independently C1-C6 alkyl that are each independently optionally substituted, optionally interrupted, and / or optionally terminated by S, O or N.

16. The method of any one of claims 10 to 15, wherein the compound is DMAPA, dimethylaminoethylamine or dimethylaminobutylamine.

17. A method of cryopreserving cells comprising: providing microparticles obtained by the method as defined in any one of claims 1 to 16; swelling the microparticles with an aqueous phase to form a jammed gel with less than 2 vol % of interstitial media, contacting the cells with the microparticles; andfreezing the cells by decreasing the temperature to -60°C or less, at a rate 0.5 - 2 degrees per minute.

18. A method of cryopreserving cells comprising combining the microparticles as obtained by the method of any one of claims 1 to 16 with cells in an aqueous jammed gel in a microparticle to cell volume ratio of 10000:1 to 1 :1 , preferably 5000:1 to 200:1 , and freezing the suspension of cells in microparticle jammed gel.

19. A cell cryopreservative comprising: a population of polymeric microparticles comprising a functionalized and hydrolyzed reaction product of a polymer comprising a structureand a cystamine crosslinker, wherein the microparticles are functionalized with a compound comprising a primary amine and a tertiary amine, and swollen into a jammed gel.

20. The cell cryopreservative of claim 19, wherein the microparticles are modified with hydrogen-bonding or cell-binding groups.

21. A process of thawing cells, comprising: providing a frozen sample comprising frozen cells and the cell cryopreservative as defined in claim 19 or 20, and separating the cell cryopreservative from the cells after thawing by diluting with a liquid cell media .

22. The process of claim 21 , further comprising sedimenting and filtering out the cell cryopreservative.

23. The process of claim 21 or 22, wherein the separating step further comprises contacting the cell cryopreservative microparticles with a reducing agent that cleaves disulfide bonds.

24. The process of any one of claims 21 to 23, wherein the frozen cells are single cells that are incorporated into the cryoprotective jammed gel.

25. The process of any one of claims 21 to 23, wherein the frozen cells are cell clusters of 10 to 3000 cells that are incorporated into the cryoprotective jammed gel.

26. The process of claim 25, wherein the cell clusters contain from 10 to 1000, or from 20 to 500 cells.

27. A method of 3D bioprinting a hydrogel structure comprising extruding a suspension of cells or cell clusters in gel formers into a jammed gel comprising an array of swollen microgel particles as obtained by the method of any one of claims 1 to 16 to obtain the hydrogel structure.

28. The method of claim 27, wherein the cell suspension in gel formers also comprises microgel particles which are formed according to the method of any one of claims 1 to 10.

29. The method of claim 27 or 28, further comprising cryopreserving the microgel structure containing cells, by performing slow freezing.

30. The method of claim 29, wherein the microgels are further loaded with proteins to produce antigens.

31. The method of any one of claims 27 to 30, wherein the support bath further comprises a viscosity enhancer selected from hyaluronic acid, carboxypropyl cellulose, carboxymethylcellulose, hydroxyethyl methacrylate, polyethylene glycol, synthetic disulfide-containing polymers and combinations thereof.

32. Use of the microgels as obtained by the method of claim 30 to deliver an antigen dose suitable to effect a vaccination.

33. The use of claim 32 wherein the microgels degrade by reductive cleavage, or hydrolysis, or a combination of reductive cleavage and hydrolysis, over time.

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