# US 2020/0399428 A1 · Systems, methods and hydrogels for cell culture and analysis > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2020/0399428 A1 - Document ID: US20200399428A1 - Status: Published application - Kind: A1 - Application number: 16/645,091 - Filed: 2018-09-11 - Published: 2020-12-24 - Earliest claimed: 2017-09-11 - Assignees: Evorion Biotechnologies GmbH - Applicants: Evorion Biotechnologies GmbH - Inventors: Hans KLEINE-BRÜGGENEY; Robert WEINGARTEN; Sebastian BÜHREN - Web page: https://patentgrep.com/patent/US20200399428A1 ## CPC - B01L 3/502738: {characterised by integrated valves (throttle valves in microfluidic sample containers B01L3/502746)} - B01L 3/502784: {specially adapted for droplet or plug flow, e.g. digital microfluidics} - C07K 14/003: {Peptide-nucleic acids (PNAs)} - C08G 73/0233: {Polyamines derived from (poly)oxazolines, (poly)oxazines or having pendant acyl groups} - C08G 81/028: {containing polyamide sequences} - C08G 83/002: {Dendritic macromolecules} - C08J 3/075: Macromolecular gels - C08J 11/105: {by treatment with enzymes} - C08L 79/02: Polyamines - C12M 25/14: {Scaffolds; Matrices (in general C12N5/0068)} - F16K 99/0015: {Diaphragm or membrane valves} - F16K 99/0051: {using electrostatic means} - F16K 99/0055: {actuated by fluids} - B01L 2200/0647: Handling flowable solids, e.g. microscopic beads, cells, particles - B01L 2200/0673: Handling of plugs of fluid surrounded by immiscible fluid - B01L 2300/0887: Laminated structure - B01L 2300/123: Flexible; Elastomeric - B01L 2400/0487: fluid pressure, pneumatics - B01L 2400/0638: membrane valves, flap valves - B01L 2400/0655: pinch valves - C08G 2210/00: Compositions for preparing hydrogels - C08J 2379/02: Polyamines - C08L 2203/02: for biomedical use - C08L 2207/53: Core-shell polymer - F16K 99/0042: {Electric operating means therefor} - B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - C08G: MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS - C08J: WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H (working, e.g. shaping, of plastics B29) - C08L: COMPOSITIONS OF MACROMOLECULAR COMPOUNDS (compositions based on polymerisable monomers C08F, C08G; artificial filaments or fibres D01F; textile treating compositions D06) - C12M: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; {APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS} - F16K: VALVES; TAPS; COCKS; ACTUATING-FLOATS; DEVICES FOR VENTING OR AERATING {(devices for emptying and evacuating the excess liquid in valves or conduits F16L55/07)} ## Abstract The present invention relates to hydrogels and polymers suitable as building blocks for hydrogels as well as advantageous methods for encapsulating cells and/or particles. Also provided are kits and methods for producing the hydrogels. ## Claims ### Claim 1 A hydrogel which comprises cross-linked hydrogel precursor molecules of the same type or of different types. ### Claim 2 (depends on 1) The hydrogel according to claim 1, wherein the hydrogel is composed of at least two different polymers with different structures as hydrogel precursor molecules, wherein optionally, at least one polymer is a copolymer. ### Claim 3 (depends on 1, 2) The hydrogel according to claim 1 or 2, wherein at least one polymer has a linear structure and at least one polymer has a multiarm or star-shaped structure. ### Claim 4 The hydrogel according to any one of the preceding claims, comprising a polymer that was obtained by copolymerization of (i) a heterocyclic chemical compound, preferably a 2-oxazoline, and (ii) a compound comprising (aa) an unsaturated imide, preferably 3-(maleimido)-propionic acid N-hydroxysuccinimide ester or (bb) an alkenyl group such as an isopropenyl group. ### Claim 5 (depends on 4) The hydrogel according to claim 4, having at least one of the following characteristics: (a) compound (ii) comprises a spacer and a functional group for crosslinking a biologically active molecule; (b) compound (ii) is a 3-(maleimido)-propionic acid N-hydroxysuccinimide ester; (c) the backbone of at least one polymer is functionalized with at least one biologically active molecule at the functional group of compound (ii). ### Claim 6 (depends on 4, 5) The hydrogel according to claim 4 or 5, wherein compound (i) is a hydrophilic poly-(2-oxazoline), wherein optionally, the water-solubility is adjusted by the 2-substitution of the 2-oxazoline compound. ### Claim 7 The hydrogel according to any of the preceding claims, wherein the backbone of the polymers is formed by hydrophilic peptide-like polymers that are crosslinked in the hydrogel by cell-compatible crosslinking reactions. ### Claim 8 The hydrogel according to any of the preceding claims, wherein the hydrogel comprises a 2-oxazoline-based polymer, preferably a poly-2-methyl-2-oxazoline based polymer, more preferably a copolymer. ### Claim 9 The hydrogel according to any of the preceding claims, wherein the hydrogel comprises (i) linear and (ii) multiarm 2-oxazoline-based polymers. ### Claim 10 (depends on 8, 9) The hydrogel according to claim 8 or 9, wherein the 2-oxazoline is substituted only at position 2 and wherein preferably, the substitution in the 2-position comprises a group selected from alkynes, alkenes, protected amine groups or short aliphatic chains such as methyl. ### Claim 11 (depends on 8, 9, 10, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155) The hydrogel according to any one of claims 8 to 10, having one or more of the following characteristics: (a) the hydrogel comprises a polymer that is formed by living cationic ring-opening polymerization of oxazolines substituted at position 2; (b) the hydrogel is a biomaterial for cell applications, wherein preferably, the biomaterial is composed of at least two different polymers according to any one of claims 101 to 155, wherein the different polymers having different structures, wherein the first polymer has a linear structure and the second polymer has a multiarm or star-shaped structure; (c) the hydrogel comprises one or more biologically active molecules linked to the polymer backbone of at least one polymer/hydrogel precursor, wherein preferably, the polymer is linear and wherein more preferably, the biologically active molecule is attached via a degradable linker. ### Claim 12 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155) The hydrogel according to any one of claims 1 to 11, wherein the hydrogel matrix is composed of a mixture of at least two different polymers according to any one of claims 101 to 155. ### Claim 13 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 101, 112, 123, 125, 139, 144) The hydrogel according to any one of claims 1 to 12, wherein the hydrogel matrix comprises at least two polymers according to: a) claim 101 or 112, wherein the polymer further has the features of claim 139, or according to claim 123; and b) claim 101 or 112, wherein the polymer further has the features of claims 139 and 144, or according to claim 125. ### Claim 14 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 101, 112, 127, 129) The hydrogel according to any one of claims 1 to 12, wherein the hydrogel matrix comprises at least two polymers according to: a) claim 101 or 112, or according to claim 127; and b) claim 101 or 112, or according to claim 129. ### Claim 15 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 124, 126) The hydrogel according to any one of claims 1 to 12, wherein the hydrogel matrix comprises at least two polymers according to: a) claim 124; and b) claim 126. ### Claim 16 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 104, 122, 123, 124, 125, 126, 127, 128, 129, 130, 134, 142, 144) The hydrogel according to any one of claims 1 to 12, wherein the hydrogel matrix comprises at least two polymers according to: a) claim 104 or 122, wherein the polymer further has the features of claim 142, wherein the polymer preferentially has a linear structure; and b) claim 104 or 122, wherein the polymer further has the features of claim 144 or wherein the hydrogel matrix comprises at least two different polymers, preferably at least 4 different polymers, more preferably at least 5 different polymers according to: a. claim 123; b. claim 125; c. claim 127; d. claim 129; e. claim 124; f. claim 126; g. claim 128; h. claim 130; and/or i. claim 134. ### Claim 17 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) A hydrogel according to any one of claims 1 to 16, wherein hydrogel precursor molecules are crosslinked in the hydrogel matrix by cell-compatible crosslinking reactions. ### Claim 18 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17) The hydrogel according to any one of claims 1 to 17, wherein the hydrogel precursor molecules are cross-linked in the hydrogel matrix by a reaction selected from: (i) a covalent bond formation, preferably selected from (aa) enzymatically catalyzed reactions, such as reactions catalyzed with transglutaminase factor XIIIa, (bb) not-enzymatically catalyzed reactions, such as click chemistry or photo-catalyzed reactions and/or (cc) uncatalyzed reactions, such as copper-free highly selective click chemistry, Michael-type addition or Diels-Alder conjugation; (ii) non-covalent bond formation preferably selected from (aa) hydrogen bonds, preferably formed by nucleic acids or nucleic acid analogs, (bb) hydrophobic interactions, (cc) Van-der-Waals interactions and (dd) electrostatic interactions; and (iii) combinations of the foregoing. ### Claim 19 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18) The hydrogel according to any one of claims 1 to 18, wherein in the hydrogel, cross-links are formed via terminating moieties that are located at ends of the polymers providing the hydrogel precursor molecules, wherein optionally, cross-links are formed exclusively via terminating moieties that are located at ends of the polymers providing the hydrogel precursor molecules. ### Claim 20 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155) The hydrogel according to any one of claims 1 to 19, having one or more of the following characteristics: (a) the hydrogel is composed of at least two different polymers, preferably according to any one of claims 101 to 155, wherein the different polymers are crosslinked with a carboxy-, thiol-, or amine-functionalized polymer, preferably polyethylene glycol (PEG) such as poly(ethylene glycol) bis(amine) or poly(ethylene glycol) dithiol or di(N-succinimidyl) functionalized components with dithiol moieties such as dithiodipropionic acid di(N-hydroxysuccinimide ester or carboxy-functionalized disulfides such as 2-carboxyethyl disulfide; (b) in the hydrogel, maleimide and thiol end functionalized polymer precursors are cross-linked, wherein preferably, at least one polymer precursor furthermore comprises a NHS ester as functional group attached to the polymeric backbone, preferably via a degradable linker, for functionalization with a biologically active molecule or wherein a biologically active molecule is attached thereto. ### Claim 21 (depends on 17, 18, 19, 20) The hydrogel according to any one of claims 17 to 20, wherein crosslinking includes hydrogen bond formation, preferably based on hybridization. ### Claim 22 (depends on 17, 18, 19, 20, 21) The hydrogel according to any one of claims 17 to 21, wherein each hydrogel precursor molecule comprises a terminating moiety and wherein terminating moieties of different hydrogel precursor molecules are crosslinked by hybridization thereby forming the hydrogel. ### Claim 23 (depends on 21, 22) The hydrogel according to any one of claims 21 to 22, wherein hybridization based cross-links are formed between (i) a linear hydrogel precursor molecule and (ii) a multiarm or starshaped hydrogel precursor molecule. ### Claim 24 (depends on 19, 20, 21, 22, 23) The hydrogel according to any one of claims 19 to 23, wherein each terminating moiety comprises bases allowing sequence specific base pairing by hydrogen bonds, in particular selected from purine and pyrimidine bases. ### Claim 25 (depends on 19, 20, 21, 22, 23, 24) The hydrogel according to any one of claims 19 to 24, having one or more of the following characteristics: (a) wherein each terminating moiety comprises an oligomer, preferably having ≤30, ≤25, preferably ≤20 or ≤15 bases, (b) the hydrogel precursors are crosslinked based on hybridization involving Watson-Crick base pairing and/or Hoogsteen base pairing, wherein preferably hybridization is based on Watson-Crick base pairing. ### Claim 26 (depends on 24, 25) The hydrogel according to claim 24 or 25, wherein the terminating moiety comprises a nucleic acid or nucleic acid analog, preferably selected from PNA, LNA, hexitol nucleic acid (HNA), morpholino oligomers, phosphorthioate DNA, phosphoramidate DNA and 2′O-methoxyethyl RNA, more preferably selected from PNA and LNA and most preferably is a PNA. ### Claim 27 (depends on 19, 20, 21, 22, 23, 24, 25, 26) The hydrogel according to any one of claims 19 to 26, wherein different hydrogel precursor molecules comprise different terminating moieties, wherein said different terminating moieties comprise moieties, preferably nucleic acids or nucleic acid analogs, that are (i) complementary to each other and are hybridized to each other in the hydrogel; or (ii) are not complementary to each other and the crosslink in the hydrogel is established by a hybridizing molecule that hybridizes to the different terminating moieties of the hydrogel precursor molecules, thereby providing a cross-link that is based on hybridization, whereby different precursor molecules are cross-linked due to hybridization, thereby providing an alternating structure of polymers, preferably linear and multimer polymers, that form the hydrogel. ### Claim 28 (depends on 27) The hydrogel according to claim 27, wherein the hybridizing molecule has one or more of the following characteristics (i) it comprises (aa) a first hybridizing portion that hybridizes to the terminating moiety of one hydrogel precursor molecule and (bb) a second hybridizing portion that hybridizes to the terminating moiety of another hydrogel precursor molecule; (ii) it comprises bases allowing sequence specific base pairing by hydrogen bonds, wherein preferably, the hybridizing molecule comprises ≤60, ≤55, preferably ≤50 or ≤35 bases. ### Claim 29 (depends on 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28) The hydrogel according to any one of claims 17 to 28, wherein the hydrogel precursor molecules are cross-linked by hybrids that comprise mismatches. ### Claim 30 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29) The hydrogel according to any one of claims 1 to 29, wherein the hydrogel precursor molecules comprise an enzyme degradable target site, preferably a protease target site, preferably located between the polymer backbone and the terminating moiety. ### Claim 31 (depends on 27, 28, 29, 30) The hydrogel according to any one of claims 27 to 30, wherein the hybridizing molecule establishing the crosslink between the terminating moieties is selected from a nucleic acid or nucleic acid analog, preferably is selected from PNA and DNA, wherein optionally, the hybridizing molecule is a PNA molecule that comprises (aa) a first hybridizing portion that hybridizes to the terminating moiety of one hydrogel precursor molecule and (bb) a second hybridizing portion that hybridizes to the terminating moiety of another hydrogel precursor molecule, and (cc) an enzyme degradable target site, preferably a protease target site, more preferably a matrix metalloprotease target site, for site directed degradation of the polymer located between the first and second hybridizing portions. ### Claim 32 (depends on 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31) The hydrogel according to any one of claims 17 to 31, wherein the crosslinking reaction includes hydrogen bond formation between two peptide nucleic acid (PNA) molecules with different base sequences or two locked nucleic acid (LNA) molecules with different base sequences or a combination of one PNA molecule and one LNA molecule. ### Claim 33 (depends on 32) The hydrogel according to claim 32, wherein a PNA molecule and/or the LNA molecule is located at the ends of the polymers that form the hydrogel. ### Claim 34 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33) The hydrogel according to any one of claims 1 to 33, wherein the hydrogel matrix is built up from precursor molecules that are cross-linked by hydrogen bonds formed by peptide nucleic acids (PNA). ### Claim 35 (depends on 34) The hydrogel according to claim 34, wherein PNA oligomers are located at the ends of the polymers that form the hydrogel. ### Claim 36 (depends on 34, 35) The hydrogel according to claim 34 or 35, wherein different precursor molecules possess complementary PNA oligomers, wherein preferably, linear and multiarm polymers are used as precursors. ### Claim 37 (depends on 34, 35, 36) The hydrogel according to any one of claims 34 to 36, wherein only different precursor molecules are cross-linked due to hybridization of the comprised PNAs, thereby providing an alternating structure of preferably linear and multimer polymers that form the hydrogel. ### Claim 38 (depends on 34, 35, 36, 37) The hydrogel according to any one of claims 34 to 37, wherein the complementary PNA oligomers possess mismatches. ### Claim 39 (depends on 34, 35, 36, 37, 38) The hydrogel according to any one of claims 34 to 38, wherein the PNA oligomers cross-linking the hydrogel precursor molecules have one or more, preferably all, of the following characteristics (i) they are short oligos ≤15 mers; (ii) they have a purine content of <50%, (iii) they are not self-complementarity, (iv) they do not comprise poly guanine sequences. ### Claim 40 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39) The hydrogel according to any one of claims 1 to 39, wherein a three-dimensional hydrogel is formed via hydrogen bonds between LNAs and/or PNAs of (i) a multiarm or starshaped poly-(2-oxazoline) based polymer and (ii) a linear poly-(2-oxazoline) based polymer, wherein preferably the linear poly-(2-oxazoline) based polymer is functionalized with biologically active molecules. ### Claim 41 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39) The hydrogel according to any one of claims 1 to 39, having one or more of the following characteristics: (a) wherein the hydrogel is formed by hybridization of complementary PNA sequences; (b) wherein precursor molecules comprise a peptide bearing a protease site, preferably adjacent to the PNA; (c) wherein the hydrogel matrix comprises multiple degradation targets for at least one enzyme that is secreted by a cell comprised in the hydrogel matrix, preferably MMP target sites; (d) wherein the hydrogel is degradable by increasing the temperature; (e) wherein the hydrogel has a spherical or plug-like structure and preferably, is spherical; (f) the hydrogel is three-dimensional; (g) the hydrogel has a size in the micrometer or sub-micrometer scale and is preferably spherical; (h) wherein the hydrogel provides a synthetic backbone, preferably a synthetic matrix that lacks toxins. ### Claim 42 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41) The hydrogel according to any one of the preceding claims 1 to 41, wherein the hydrogel matrix comprises one or more cells and/or particles, preferably comprises at least one cell. ### Claim 43 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42) The hydrogel according to any one of the preceding claims 1 to 42, wherein at least one polymer is functionalized with at least one biologically active molecule, preferably to present different adhesive ligands, bioactive compounds and functional biomolecules such as adhesive compounds of the extra cellular matrix (ECM), growth factors, antibodies, CRISPR-Cas and nucleic acids or wherein the hydrogel comprises one or more of the following: (i) functional molecules for cell culture and cell analysis; (ii) gold particles, quantum dots, growth promoting substances, cytokines, chemokines, antibody-conjugates and/or inorganic substances. ### Claim 44 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43) The hydrogel according to any one of the preceding claims 1 to 43, wherein the backbone of a comprised polymer, preferably a linear polymer, is functionalized with a biologically active molecule. ### Claim 45 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44) The hydrogel according to any one of the preceding claims 1 to 44, wherein the hydrogel comprises capture molecules, which may be incorporated by one or more of the cross-linking techniques as defined in claim 18, wherein optionally, incorporation of the capture molecules involves peptide nucleic acids, wherein preferably, a PNA oligomer is incorporated into the hydrogel gel by amide bond formation between an NHS-ester from the hydrogel precursor molecule and the primary amine of a PNA oligomer and wherein the capture molecule is fused to a complementary PNA oligomer and wherein the capture molecule is incorporated into the hydrogel by hydrogen bond formation between the two PNA oligomers. ### Claim 46 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45) The hydrogel according to any one of the preceding claims 1 to 45, wherein the hydrogel is surrounded by a gel-shell. ### Claim 47 (depends on 46) The hydrogel according to claim 46, wherein the gel-shell comprises at least one gel-shell forming compound that is optionally crosslinked to the hydrogel, wherein preferably the gel-shell forming compound is a primary amine bearing polymer molecule, e.g. a poly (allylamine). ### Claim 48 (depends on 47) The hydrogel according to claim 47, wherein the gel-shell forming compound is or is derived from a compound selected from the group consisting of poly(allylamine), branched amino-polyethyleglycol (PEG), branched polyethylenimine (PEI), polylysine, poly amidoamine (PAMAM) dendrimer, poly(β-amino ester), chitosan and poly(2-amino-2-oxazoline). ### Claim 49 (depends on 47, 48) The hydrogel according to claim 47 or 48, having one or more of the following characteristics: (a) the gel-shell forming compound is crosslinked to a functional group of a poly(2-oxazoline)copolymer); (b) the gel-shell forming compound forms a gel-shell around the hydrogel matrix, wherein the gel-shell is not covalently attached to the hydrogel matrix and wherein optionally, the gel shell forming compound comprises functional groups that are cross-linked via a further compound thereby forming a gel-shell around the hydrogel matrix; (c) the gel-shell forming compound comprises a nucleic acid or nucleic acid analog, preferably a PNA sequence, that is hybridized to another compound comprised in the hydrogel matrix, wherein the compound is a multi-arm or star-shaped polymer, which is not crosslinked to the hydrogel matrix and wherein the gel-shell forming compound is preferably a linear polymer. ### Claim 50 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 104) The hydrogel according to any one of claims 1 to 49, comprising a polymer according to claim 104 as hydrogel precursor that is preferably cross-linked with a multiarm polymer. ### Claim 51 (depends on 47, 48, 49, 50) The hydrogel according to any one of claims 47 to 50, wherein the gel-shell surrounded hydrogel was obtained by the method according to 64 to 70. ### Claim 52 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51) A method for producing a hydrogel according to any one of claims 1 to 51, wherein the hydrogel matrix is formed by cross-linking hydrogel precursor molecules of the same type or of different types. ### Claim 53 (depends on 52) The method according to claim 52, comprising crosslinking hydrogel precursor molecules by a cell-compatible crosslinking reaction, preferably in the presence of a cell. ### Claim 54 (depends on 52, 53) The method according to claim 52 or claim 53, wherein the crosslinking reaction is selected from: (i) a covalent bond formation, preferably selected from (aa) enzymatically catalyzed reactions, such as reactions catalyzed with transglutaminase factor XIIIa, (bb) not-enzymatically catalyzed reactions, such as click chemistry or photo-catalyzed reactions and/or (cc) uncatalyzed reactions, such as copper-free highly selective click chemistry, Michael-type addition or Diels-Alder conjugation; (ii) non-covalent bond formation preferably selected from (aa) hydrogen bonds, preferably formed by nucleic acids or nucleic acid analogs, (bb) hydrophobic interactions, (cc) Van-der-Waals interactions and (dd) electrostatic interactions; and (iii) combinations of the foregoing. ### Claim 55 (depends on 52, 53, 54) The method according to any one of claims 52 to 54, wherein the crosslinking reaction includes hydrogen bond formation, preferably between (i) two peptide nucleic acid (PNA) molecules with different base sequences or (ii) two locked nucleic acid (LNA) molecules with different base sequences or (iii) a combination of one PNA molecule and one LNA molecule. ### Claim 56 (depends on 52, 53, 54, 55) The method according to any one of claims 52 to 55, wherein the hydrogel is produced using a method for droplet generation and mixing of at least two droplets, preferably the method for droplet generation as disclosed herein. ### Claim 57 (depends on 56) The method according to claim 56, wherein after generating and mixing said droplets, a spherical or plug-like hydrogel matrix is formed within the mixed droplet. ### Claim 58 (depends on 56, 57) The method according to claim 56 or 57, comprising generating (i) a first droplet that comprises a multiarm hydrogel precursor, (ii) a second droplet that comprises a linear hydrogel precursor, (iii) optionally a third droplet for initiating the cross-linking of the multiarm and the linear hydrogel precursor, wherein after generating and mixing said droplets a spherical or plug-like hydrogel matrix is formed within the mixed droplet, wherein optionally, the first or the second droplet comprises compounds, preferably biological active molecules, wherein said compounds are immobilized within the hydrogel matrix, preferably during hydrogel formation. ### Claim 59 (depends on 56, 57, 58) The method according to any one of claims 56 to 58, comprising generating and mixing at least four droplets, wherein the fourth droplet comprises a compound that becomes immobilized within the formed hydrogel matrix, preferably by a stable amide bond, wherein the compounds is optionally selected from proteins such as antibodies, growth factors or ECM proteins; nucleic acids such as DNA primers and peptide nucleic acids or is selected from gold particles, quantum dots, growth promoting substances, cytokines, chemokines, antibody-conjugates, inorganic substances. ### Claim 60 (depends on 56, 57, 58, 59) The method according to any one of claims 56 to 59, wherein the droplets are generated in parallel or sequentially. ### Claim 61 (depends on 52, 53, 54, 55, 56, 57, 58, 59, 60) The method according to any one of claims 52 to 60, having one or more of the following characteristics: (a) the method comprises generating at least two droplets, wherein each droplet comprises a different cell type and fusing said at least two droplets to provide a first droplet that comprises at least one multiarm precursor; (b) the first droplet comprises at least one cell and wherein the multiarm precursor lacks functional groups that are reactive with the one or more cells under the conditions within the first droplet. ### Claim 62 (depends on 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83) The method according to any one of claims 52 to 61, wherein during hydrogel formation, one or more cells or particles, preferably at least one cell, becomes encapsulated in the hydrogel matrix, wherein preferably, the one or more cells or particles are combined with at least one hydrogel precursor prior to forming the gel, and wherein more preferably, the encapsulation method as defined in any of claims 73 to 83 is used. ### Claim 63 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62) The method according to any one of claims 52 to 62, comprising functionalizing the hydrogel with at least one biologically active molecule, wherein preferably, functionalization has one or more of the following characteristics: (a) functionalization occurs before, during or after encapsulating at least one cell into the hydrogel; (b) the biologically active molecule is cross-linked to a functional group of at least one polymer that provides a hydrogel precursor molecule, which preferably is a polymer as defined in any one of claims 1 to 55, and preferably, is a linear polymer; (c) after hydrogel formation the method comprises adding bioactive molecules to a liquid that flows through the formed hydrogel, thereby incorporating bioactive molecules into the hydrogel matrix; (d) the hydrogel is functionalized with at least one biologically active molecule before a gel-shell is formed that surrounds the particle. ### Claim 64 (depends on 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63) The method according to any one of claims 52 to 63, for producing a gel-shell surrounded hydrogel, comprising (a) providing a droplet generated by fusion of multiple droplets, wherein the fused droplet A comprises the hydrogel matrix; (b) forming the gel-shell by fusing droplet A with a second droplet B containing a polymer which comprises primary amines, such as poly allylamine polymers, thereby providing a larger droplet C containing said hydrogel matrix with the volume of the hydrogel matrix being smaller than the volume of droplet C and wherein in droplet C, said hydrogel matrix is surrounded by said polymer from droplet B and (c) crosslinking of the hydrogel polymers at the edge of the hydrogel matrix. ### Claim 65 (depends on 64) The method according to claim 64, wherein said polymer from droplet B diffuses into the hydrogel matrix, whereby crosslinking occurs. ### Claim 66 (depends on 64, 65) The method according to claim 64 or 65, wherein the method comprises using (i) a primary amine bearing polymer molecule, e.g. a poly allylamine and (ii) a small primary amine, e.g. 3-amino-1,2-propanediol, wherein the polymer molecule (i) having a smaller diffusion coefficient than the small primary amine (ii). ### Claim 67 (depends on 66) The method according to claim 66, wherein the primary amine diffuses faster into said hydrogel matrix than the polymer molecule, wherein preferably the small primary amines are added with a short delay after the poly allylamine polymers. ### Claim 68 (depends on 64, 65, 66, 67) The method according to any one of claims 64 to 67, wherein the method further comprises fusing said droplet C with a droplet D containing a small primary amine, e.g. 3-amino-1,2-propanediol, the small primary amine having a smaller diffusion coefficient than the polymer in droplet C. ### Claim 69 The method according to any one of the preceding claims for producing a gel-shell surrounded hydrogel, comprising (a) providing a droplet generated by fusion of multiple droplets, wherein the fused droplet A comprises the hydrogel matrix; (b) forming the gel-shell by immobilizing and diffusing droplet A by an aqueous phase containing a small primary amine, e.g. 3-amino-1,2-propanediol and a polymer which comprises primary amines, such as poly allylamine polymers, the small primary amine having a smaller diffusion coefficient than the polymer; and (c) crosslinking of the hydrogel polymers at the edge of the hydrogel matrix. ### Claim 70 The method according to any one of the preceding claims for producing a gel-shell surrounded hydrogel, wherein the shell is formed by contacting one or more of the following compounds with the hydrogel matrix: i) a polymer which comprises primary amines, which is preferably selected from poly(allylamine), (branched) amino-polyethyleglycol (PEG), (branched) polyethylenimine (PEI), polylysine, poly amidoamine (PAMAM) dendrimer, poly(β-amino ester), chitosan, or amino-PaOX, and, optionally, a primary amine compound which preferably is a small primary amine compound such as an aminofunctionalyzed C3-C6-alkanediol, e.g. 2-amino-1,3-propanediol or 3-amino-1,2-propanediol, wherein amine groups react with a residual functional group of the hydrogel matrix, e.g. a N-hydroxysuccinimide ester; ii) a polymer comprising a N-hydroxysuccinimide ester, preferably selected from PEG-NHS-ester or polyoxazoline-NHS-ester, and a diamine compound, e.g. a c3-c6-alkanol diamine such as 1,3-diamino-2-propanol, wherein the diamine compound is present in the hydrogel matrix prior to adding the N-hydroxysuccinimide ester comprising compound; iii) a polymer comprising a maleimide, and furthermore a dithiol compound, e.g. 2,2′-(ethylenedioxy)diethanethiol or short dithiol functionalized polymers with an enzyme degradable target site, such as a matrix metalloprotease sensitive target site, wherein the dithiol compound is present in the hydrogel matrix prior to adding the maleimide comprising compound; or iv) the gel-shell forming compound comprises a nucleic acid or nucleic acid analog, preferably a PNA sequence, that is hybridized to another compound comprised in the hydrogel matrix, wherein the compound is a multi-arm or star-shaped polymer, which is not crosslinked to the hydrogel matrix and wherein the gel-shell forming compound is preferably a linear polymer. ### Claim 71 (depends on 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151) The method according to any one of the preceding claims 52 to 70, comprising providing at least two polymers, preferably selected from the polymers as defined in any one of claims 101 to 151, as hydrogel precursors and cross-linking the at least two polymers to provide the hydrogel. ### Claim 72 (depends on 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71) A hydrogel obtained by the method according to any one of claims 52 to 71. ### Claim 73 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51) A method for encapsulating one or more cells and/or particles into a hydrogel, preferably a hydrogel as defined in any one of claims 1 to 51, wherein the one or more cells and/or particles are combined with at least one hydrogel precursor prior to gel formation and are encapsulated into the hydrogel matrix during hydrogel formation. ### Claim 74 (depends on 73) The method according to claim 73, wherein one or more polyoxazoline derivatives are used as hydrogel precursor. ### Claim 75 (depends on 74, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155) The method according to claim 74, wherein one or more polymers as defined in any one of claims 101 to 155 are used as hydrogel precursor, wherein preferably, at least one polyoxazoline based polymer, preferably a co-polymer comprising at least one moiety of formula (I) and at least one moiety of formula (II) as defined in claim 101 and claims dependent thereon, is used as hydrogel precursor. ### Claim 76 (depends on 73, 74, 75) The method according to any one of claims 73 to 75, having one or more of the following characteristics: (a) wherein after encapsulation, the one or more cells are in the center of the hydrogel, (b) the method comprises preparing a liquid composition comprising (i) one or more cells and/or particles, preferably one or more cells, and (ii) the hydrogel precursors, and cross-linking the hydrogel precursors thereby providing a hydrogel encapsulating the one or more cells and/or particles. (c) the method comprises preparing a liquid composition using a microfabricated valve as disclosed herein comprising (i) one or more cells and/or particles, preferably one or more cells, and (ii) the hydrogel precursors, and cross-linking the hydrogel precursors thereby providing a hydrogel encapsulating the one or more cells and/or particles. ### Claim 77 (depends on 76) The method according to claim 76, wherein the method comprises combining one or more cells and/or particles, preferably one or more cells, with at least one hydrogel precursor prior to forming the hydrogel and wherein the hydrogel is formed in the presence of the one or more cells. ### Claim 78 (depends on 73, 77) The method according to claim 73 or 77, wherein the method comprises (a) providing prior to forming the hydrogel a liquid composition, e.g. in form of a droplet, wherein the composition comprises (i) one or more cells and/or particles, preferably one or more cells, and (ii) at least one hydrogel precursor, (b) combining, e.g. mixing, said composition with at least one further hydrogel precursor and (c) forming the hydrogel in the presence of the one or more cells by cross-linking (gelation) whereby the hydrogel matrix is formed around the cell. ### Claim 79 (depends on 73, 74, 75, 76, 77, 78) The method according to any one of claims 73 to 78, comprising combining the one or more cells sequentially with the hydrogel precursors prior to cross-linking the hydrogel precursors, wherein the one or more cells are combined with at least one hydrogel precursor that lacks functional groups that are reactive with the one or more cells under the combination conditions and subsequently adding at least one further hydrogel precursor, wherein optionally, the subsequently added hydrogel precursor comprises functional groups for attaching a biologically active molecule. ### Claim 80 (depends on 56, 73, 74, 75, 76, 77, 78, 79) The method according to any one of claims 73 to 79, wherein the hydrogel is produced by generating and mixing at least two droplets comprising different hydrogel precursors, wherein one or more cells are comprised in at least one droplet, preferably using the method for droplet generation and fusing of at least two droplets as defined in any one of preceding claims or using the method according to claim 56 and claims dependent thereon. ### Claim 81 (depends on 73, 74, 75, 76, 77, 78, 79, 80) The method according to any one of claims 73 to 80, wherein the method comprises: encapsulating one or more cells and/or particles into a first droplet, wherein the first droplet has a defined size and comprises a hydrogel precursor molecule (a) at a defined concentration; generating a second droplet, wherein the second droplet has a defined size and comprises a hydrogel precursor molecule (b) at a defined concentration fusing said formed droplets, thereby providing a larger droplet that contains the hydrogel precursor molecules (a) and (b) and the one or more cells and/or particles, wherein preferably, hydrogel formation occurs due to the mixing of said hydrogel precursor molecules. ### Claim 82 (depends on 73, 74, 75, 76, 77, 78, 79, 80, 81) The method according to any one of claims 73 to 81, wherein the first or the second droplet comprises compounds, preferably biological active molecules, wherein said compounds are immobilized within the hydrogel matrix, preferably during hydrogel formation. ### Claim 83 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82) The method according to any one of claims 73 to 82, having one or more of the following characteristics (a) the hydrogel is as defined in any one of claims 1 to 51; (b) wherein the type of encapsulated cells is the same or different; (c) wherein the method comprises preparing at least two separate hydrogels, preferably at least two hydrogel beads, wherein the type of encapsulated cells is the same or different. ### Claim 84 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51) A method for degrading a hydrogel according to any one of claims 1 to 51, comprising reversing the cross-links of the hydrogel. ### Claim 85 (depends on 84) The method according to claim 84, comprising reversing hybridization based, preferably PNA based, cross-links of the hydrogel. ### Claim 86 (depends on 84, 85) The method according to claim 84 or 85, comprising one or more of the following: (a) heating the hydrogel to degrade the hydrogel; and/or (b) increasing the ionic strength; (c) dehybridization of complementary PNAs by applying heat, high salt concentrations or complementary nucleic acids with a higher affinity, preferably in molar excess. ### Claim 87 (depends on 84, 85, 86) The method according to any one of claims 84 to 86, comprising adding at least one hybridizing molecule, preferably in excess, to the hydrogel wherein the hybridizing molecule disturbs the cross-linking hybrids of the hydrogel, whereby the crosslinks are reversed. ### Claim 88 (depends on 87) The method according to claim 87, wherein the at least one hybridizing molecule used for degradation is complementary to (aa) a terminating moiety of a hydrogel precursor molecule that participates in the crosslinking hybrid, or (bb) the hybridizing molecule used for crosslinking according to claim (ii), and binds with a higher affinity thereto. ### Claim 89 (depends on 87, 88) The method according to claim 87 or 88, wherein the crosslinking hybrid comprises mismatches and wherein the added hybridizing molecule provides a hybrid without mismatches. ### Claim 90 (depends on 87, 88, 89) The method according to any one of claims 87 to 89, wherein the added hybridizing molecule is selected from a nucleic acid or nucleic acid analog, and preferably is a PNA. ### Claim 91 (depends on 85) The method according to claim 85, comprising adding PNA oligomers in excess to the hydrogel, wherein the complementary PNAs forming the cross-link of the hydrogel have a decreased hybridization energy compared to the PNA oligomers added for hydrogel degradation. ### Claim 92 (depends on 84, 85, 86, 87, 88, 89, 90, 91) The method according to any one of claims 84 to 91, comprising adding at least one enzyme to degrade the hydrogel, preferably selected from proteases and nucleases. ### Claim 93 (depends on 92) The method according to claim 92, wherein the added enzyme targets a protease target site comprised in the hydrogel precursor and/or the hybridizing molecule, thereby degrading the hydrogel. ### Claim 94 (depends on 92, 93) The method according to claim 92 or claim 93, wherein the added enzyme is a nuclease, preferably a DNase, and wherein the DNase degrades a hybridizing DNA molecule that establishes the hybridizing hybrid thereby degrading the hydrogel. ### Claim 95 (depends on 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94) The method according to any one of the preceding claims 84 to 94, wherein the hydrogel to be degraded comprises at least one cell and wherein said cell is not affected by the degradation procedure. ### Claim 96 (depends on 95) The method according to claim 95, wherein the hydrogel is degraded by at least one enzyme that is secreted by the at least one cell comprised in the hydrogel, wherein optionally (a) the secreted enzyme targets the protease target site comprised in the hydrogel precursor and/or the hybridizing molecule, thereby degrading the hydrogel; (b) the secreted enzyme is a nuclease, preferably a DNase, and wherein the DNase degrades the hybridizing DNA molecule that establishes the hybridizing hybrid thereby degrading the hydrogel. ### Claim 97 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52) A droplet or combination of at least two droplets comprising a hydrogel according to any one of claims 1 to 52. ### Claim 98 (depends on 97) The droplet according to claim 97, wherein the droplet or the combination of at least two droplets comprises one or more cells, wherein optionally, at least two different cell types are comprised, preferably in different droplets. ### Claim 99 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51) A kit for providing a hydrogel according to any one of claims 1 to 51, comprising: (a) a first hydrogel precursor (b) a second hydrogel precursor (c) optionally a reagent for crosslinking the first and second hydrogel precursor (d) optionally a test device as disclosed herein. ### Claim 100 (depends on 99, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155) The kit according to claim 99, having one or more of the following characteristics: the first and second hydrogel precursor are provided by a polymer as defined in any one of claims 101 to 155, the first and second hydrogel precursor comprise terminating moieties comprising nucleic acids or nucleic acid analogs; the first hydrogel precursor is a linear polymer and the second hydrogel precursor is a multiarm or starshaped polymer; the first and/or the second hydrogel precursor, preferably the first and second hydrogel precursor, is selected from a polymer as defined in any one of 101 to 155; it comprises at least one biologically active molecule, wherein preferably, said molecule is suitable to react with a functional group of at least one hydrogel precursor, preferably a linear hydrogel precursor, and wherein more preferably, the biologically active molecule is a peptide or protein and wherein at least one hydrogel precursor comprises a functional group capable of reacting with the N-terminus of the peptide or protein, wherein preferably, the functional group of the hydrogel precursor is a NHS ester; it comprises a reagent for providing a gel shell; it comprises a cell culture medium; the hydrogel precursor molecules are lyophilized, wherein optionally the kit comprises a reagent for reconstituting the hydrogel precursor molecules. ### Claim 101 Polymer, especially polymer as building-block for hydrogel formation, comprising at least one moiety of formula (I) and at least one moiety of formula (II) wherein R1 is a hydrogen atom, a hydrocarbon with 1-18 carbonatoms (preferably CH3, —C2H5), a C1-C25-hydrocarbon with at least one hydroxy group, a C1-C25-hydrocarbon with at least one carboxy group, (C2-C6)alkylthiol, (C2-C6)alkylamine, protected (C2-C6)alkylamine (preferably-(CH2)2-6—NH—CO—R (with R=benzylhydryloxy, 9-fluorenylmethoxy)), (C2-C6)alkylazide, polyethylene glycol, a crosslink to R1 of another moiety of formula (I), polylactic acid, polyglycolic acid or polyoxazoline, or wherein R1 is a residue R4, R2 and R3 R2 and R3 are linked to form a cyclic moiety of formula (II) comprising at least one residue R4 or R2 and R3 are independently selected from hydrogen, —COOH, methyl or a residue R4, wherein optionally, at least one of R2 and R3 is a residue R4, R4 is a moiety, comprising at least one functional group, independently selected from a functional group for crosslinking and/or for binding biologically active compounds, and optionally comprising a (preferably degradable) spacer moiety connecting said functional group with the binding site of the respective moiety of formula (I) or formula (II), and R5 denotes a hydrogen atom, a carboxymethyl group or a methyl group, x is 1, 2 or 3, and * denotes a chemical bond of the polymer backbone or to a terminating moiety, with the proviso, that at least one moiety of formula (I) or formula (II) comprises a residue R4, wherein preferably only the moieties of formula (I) or only the moieties of formula (II) comprise at least one moiety R4. ### Claim 102 (depends on 101) Polymer, especially polymer as building-block for hydrogel formation, according to claim 101, characterized in, that R1 is a hydrogen atom or a C1-C18-alkyl group, preferably a hydrogen atom, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, iso-pentyl, neopentyl, sec-pentyl, hexyl, heptyl, octyl, nonyl or decyl, more preferably methyl or ethyl. ### Claim 103 (depends on 101, 102) Polymer, especially polymer as building-block for hydrogel formation, according to claim 101 or claim 102, characterized in, that it comprises at least two different moieties of formula (I) having different groups R1. ### Claim 104 Polymer, especially polymer as building-block for hydrogel formation, according to any of the preceding claims, characterized in that: R1 is a hydrogen atom or a hydrocarbon with 1-18 carbon atoms, preferably for adjusting chemical characteristics of the polymer; R2 and R3 are linked to form a cyclic moiety of formula (II) comprising at least one N-hydroxysuccinimide ester for binding biologically active compounds or R2 and R3 are independently selected from hydrogen, —COOH, methyl or at least N-hydroxsuccinimide bearing molecule for binding biologically active compounds; R5 denotes a hydrogen atom, a carboxymethyl group or a methyl group; x is 1; and * denotes a chemical bond of the polymer backbone or to a terminating moiety wherein the terminating moiety preferably comprises a PNA sequence. ### Claim 105 Polymer, especially polymer as building-block for hydrogel formation, according to any of the preceding claims, characterized in, that x is 1 or 2, preferably x is 1. ### Claim 106 Polymer, especially polymer as building-block for hydrogel formation, according to any of the preceding claims, characterized in, that it comprises at least one moiety of formula (II), selected from a moiety of formula (II-a) wherein R4 is a moiety comprising at least one functional group for crosslinking and/or for binding biologically active compounds, and optionally comprising a (preferably degradable) spacer moiety connecting said functional group with the binding site of R4 according to formula (II-a), and and * denotes a chemical bond of the polymer backbone or to a terminating moiety. ### Claim 107 Polymer, especially polymer as building-block for hydrogel formation, according to any one of the preceding claims, characterized in, that it comprises at least one moiety of formula (II), selected from a moiety of formula (II-b) wherein R5 and R4 is defined according to any of the preceding claims, R2 is a hydrogen atom or a carboxyl group, Q denotes an oxygen atom or an imino group NH, and * denotes a chemical bond of the polymer backbone or to a terminating moiety. ### Claim 108 Polymer, especially polymer as building-block for hydrogel formation, according to any of the preceding claims, characterized in, that R4 is independently a moiety, comprising at least one functional group independently selected from arene, amine, alkyne, azide, anhydride, acid anhydride, ketone, haloalkane, imidoester, diol, hemiacetal, acrylate, alkene, thiol, ether, ester, isocyanate, isothiocyanate, succinimide, N-hydroxysuccinimide, sulfo-N-hydroxysuccinimide, amide, maleimide, N-heterocyclic carbene, acyl halide, N-heterocyclic phosphine, hydrazide, nitrile, aminoxy, imidazolide, imine, aldehyde, azo compound, imide, carbodiimide, haloacetyl, pyridyl disulfide, carboxamide, vinyl ether, carboxyl, carboxylate, phenyl, phenol, indol, methylthiol, pyridyldithiol, hydroxyl, epoxide, carbonyl, methoxycarbonyl, glycidyl, carboxyphenyl. ### Claim 109 Polymer, especially polymer as building-block for hydrogel formation, according to any of the preceding claims, characterized in, that the moiety of the formula (II) is derived from at least one monomer selected from an unsaturated imide (preferably derived from maleimide), an alkene, an acrylic acid, an itaconic acid, a lactone (preferably β-propiolactone, α-methyl-β-propiolactone, α,α-dimethyl β-propiolactone, β-butyrolactone), an acrylamide, a sulfonamide (preferably ethylensulfonamide), an anhydride, a methacrylic acid, an acrylamide, a methacrylamide, a N,N-diacrylamide (preferably N-methyldiacrylamide), a 1-propanesulfonic acid sultone, with the proviso, that said monomer comprise said residue R4 respectively. ### Claim 110 Polymer, especially polymer as building-block for hydrogel formation, according to any of the preceding claims, characterized in, that said functional group of residue R4 is independently selected from the group consisting of protected N-hydroxysuccinimide-esters, unprotected N-hydroxysuccinimide-esters, sulfo-N-hydroxysuccinimide esters, vinyl sulfone, sulfonyl chloride, aldehyde, epoxides, thiol, maleimide and carbonate, wherein preferably, the moiety of formula (II) comprises such residue R4. ### Claim 111 Polymer, especially polymer as building-block for hydrogel formation, according to any of the preceding claims, characterized in, that the moiety of the formula (II) is derived from monomers selected from 3-(maleimido)-propionic acid N-hydroxysuccinimide ester, 6-Maleimidohexanoic acid N-hydroxysuccinimide ester, N-(Methacryloxy)-succinimideisopropenyl, BMPH (N-(β-maleimidopropionic acid)-hydrazide, EMCH (N-ε-maleimidocaproic acid hydrazide), PDPH (3-(2-pyridyldithio) propionyl hydrazide), Methacrylic acid N-hydroxysuccinimide ester, N-methoxycarbonyl maleimide, acrylic acid N-hydroxysuccinimide ester, a PNA-amide of acrylic acid, a PNA-amide of methacrylic acid, a PNA-amide of acrylamide, a PNA-amide of methacrylamide, a monomer of formula wherein n is an integer of at least 1, a monomer of formula wherein n is an integer of at least 1, a monomer of formula, wherein n is an integer greater than 1 and Base is independently a moiety comprising at least one nucleobase, or mixtures thereof. ### Claim 112 (depends on 101) Polymer, especially polymer as building-block for hydrogel formation, characterized in, that it comprises at least one (m is an integer of at least 1) unit having the structure of formula (III) R2 is independently a residue R4, comprising at least one functional group for crosslinking and/or for binding biologically active compounds, S1 is independently defined according to R1 of claim 101, fragment D-Cn is part of the polymer backbone, wherein said structure results from polymerization of a heterocyclic molecule B in presence of a first component A. ### Claim 113 (depends on 112) Polymer, especially polymer as building-block for hydrogel formation, according to claim 112, characterized in, that said first component A is a compound of formula (IV) R1-k-R2 (IV) wherein R1 is a first functional group for the copolymerization with said heterocyclic molecule B, R2 is said moiety R4, k is a direct bond or a spacer. ### Claim 114 (depends on 112, 113) Polymer, especially polymer as building-block for hydrogel formation, according to claim 112 or claim 113, characterized in, that k is selected from a direct bond, alkylidene groups with 2 to 8 carbon atoms, hydrocarbons, and/or a degradable spacer (preferably selected from peptides, PNA, polyethylene glycol). ### Claim 115 (depends on 109, 110, 111, 112, 113, 114) Polymer, especially polymer as building-block for hydrogel formation, according to any of the claims 112 to 114, characterized in, that said first component A of formula (IV) is selected from the monomers as defined in any of the claims 109 to 111. ### Claim 116 (depends on 1, 112, 113, 114, 115) Polymer, especially polymer as building-block for hydrogel formation, according to any of the claims 112 to 115, characterized in, that said heterocyclic molecule B is a 2-substituted heterocyclic compound of formula (V) D-S1 (V) wherein D is an oxazoline-moiety, oxazine-moiety or oxyazepine-moiety and S1 is a substituent in 2-position as defined as R1 of claim 1. ### Claim 117 (depends on 112, 113, 114, 115, 116) Polymer, especially polymer as building-block for hydrogel formation, according to any of the claims 112 to 116, characterized in, that said unit is a covalently functionalized D-substituted alkylamine. ### Claim 118 (depends on 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117) Polymer, especially polymer as building-block for hydrogel formation, according to any of the claims 112 to 117, characterized in, that it is a polymer according to any claim of claims 101 to 111. ### Claim 119 Polymer, especially polymer as building-block for hydrogel formation, of formula (P1) wherein R is independently selected from a hydrogen atom, a hydrocarbon with 1-18 carbonatoms (preferably CH3, —C2H5), a C1-C25-hydrocarbon with at least one hydroxy group, a C1-C25-hydrocarbon with at least one carboxy group, (C2-C6)alkylthiol, (C2-C6)alkylamine, protected (C2-C6)alkylamine (preferably-(CH2)2-6—NH—CO—R (with R=tert-Butyl, perfluoroalkyl)), (C2-C6)alkylazide, polyethylene glycol, polylactic acid, polyglycolic acid, polyoxazoline, or wherein R is a residue R4 Y is a moiety containing at least one graft, comprising at least one residue R4, T1 is a terminating moiety, which may contain a residue R4, T2 is a terminating moiety, which contains a residue R4, p is an integer from 1 to 10, n is an integer greater than 1 and preferably, below 500, m is zero or an integer of at least, preferably greater than 1, and preferably, below 500, the sum n+m is greater than 10, x is independently 1, 2 or 3, preferably x is independently 1 or 2, most preferably x is 1, R4 independently comprise at least one functional group for crosslinking and/or for binding biologically active compounds, and optionally comprising a (preferably degradable) spacer moiety connecting said functional group with the binding site to the respective moiety of the structure of formula (P1), wherein the entirety of all m-fold and n-fold repeating units are distributed in any order within the polymer chain and wherein optionally, the polymer is a random copolymer or a block copolymer. ### Claim 120 (depends on 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 119) Polymer, especially polymer as building-block for hydrogel formation, of claim 119 characterized in, that Y is a moiety of formula (II) as defined in any of the claims 101 to 111. ### Claim 121 (depends on 119, 120) Polymer, especially polymer as building-block for hydrogel formation, of claim 119 or claim 120, characterized in, that R is a hydrogen atom or a C1-C18-alkyl group, (preferably a hydrogen atom, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, iso-pentyl, neopentyl, sec-pentyl, hexyl, heptyl, octyl, nonyl, decyl) and m is an integer greater than 1. ### Claim 122 (depends on 119, 120, 121) Polymer, especially polymer as building-block for hydrogel formation, of any of claims 119 to 121, characterized in, that R is a hydrogen atom, a hydrocarbon with 1-18 carbonatoms (preferably CH3, —C2H5); Y is a moiety containing at least one graft, comprising at least one degradable spacer moiety connecting at least one N-hydroxysuccinimide ester for binding biologically active compounds to the respective moiety of the structure of formula (P1); T1 is a terminating moiety, optionally comprising a peptide nucleic acid (PNA) sequence; T2 is a terminating moiety, optionally comprising a peptide nucleic acid (PNA) sequence; n is an integer greater than 1; m is an integer greater than 1; the sum n+m is greater than 10 and less than 500; and x is 1; wherein the entirety of all m-fold and n-fold repeating units are distributed in any order within the polymer chain and wherein optionally, the polymer is a random copolymer or a block copolymer. ### Claim 123 (depends on 119, 120, 121) Polymer, especially polymer as building-block for hydrogel formation, according to any of the claims 119 to 121, characterized in, that T1 is a terminating moiety, comprising a first XNA-residue (XNA1) and optionally a EDTS-moiety, T2 is a terminating moiety, comprising a second XNA-residue (XNA2) and optionally a EDTS-moiety, p equals 1 or 2, preferably equals 1, EDTS is an enzyme degradable target site, preferably a matrix metalloprotease (MMP) target site, for site directed degradation of the polymer, XNA is a nucleic acid or nucleic acid analog, preferably a peptide nucleic acid (PNA) sequence. ### Claim 124 (depends on 123) Polymer, especially polymer as building-block for hydrogel formation, of claim 123, characterized in, that m is zero and no moiety Y is comprised in the polymer. ### Claim 125 (depends on 119, 120, 121) Polymer, especially polymer as building-block for hydrogel formation, according to any of the claims 119 to 121, characterized in, that T1 is a terminating moiety, comprising no residue R4, T2 is a terminating moiety, comprising a XNA-residue, optionally linked to a EDTS-moiety, p is an integer of 3 to 10, preferably 3 to 10, preferably 3 to 8, most preferred 3 to 6, EDTS is an enzyme degradable target site, preferably a matrix metalloprotease (MMP) target site, for site directed degradation of the polymer, XNA is a nucleic acid or nucleic acid analog, preferably a peptide nucleic acid (PNA) sequence. ### Claim 126 (depends on 125) Polymer, especially polymer as building-block for hydrogel formation, of claim 125, characterized in, that m is zero and no moiety Y is comprised in the polymer. ### Claim 127 (depends on 119, 120, 121) Polymer, especially polymer as building-block for hydrogel formation, according to any of the claims 119 to 121, characterized in, that T1 is a terminating moiety, comprising a residue R4 different from a XNA-residue, wherein R4 is optionally linked to a EDTS-moiety, T2 is a terminating moiety, comprising a residue R4 different from a XNA-residue, wherein R4 is optionally linked to an EDTS-moiety, p equals 1 or 2, preferably equals 1, EDTS is an enzyme degradable target site, preferably a matrix metalloprotease (MMP) target site, for site directed degradation of the polymer, XNA is a nucleic acid or nucleic acid analog, preferably a peptide nucleic acid (PNA) sequence. ### Claim 128 (depends on 127) Polymer, especially polymer as building-block for hydrogel formation, of claim 127, characterized in, that m is zero and no moiety Y is comprised in the polymer. ### Claim 129 (depends on 119, 120, 121) Polymer, especially polymer as building-block for hydrogel formation, according to any of the claims 119 to 121, characterized in, that T1 is a terminating moiety, comprising no residue R4, T2 is a terminating moiety, comprising a residue R4 different from a XNA-residue, wherein R4 is optionally linked to an EDTS-moiety, p is an integer of 3 to 10, preferably 3 to 10, preferably 3 to 8, most preferred 3 to 6, EDTS is an enzyme degradable target site, preferably a matrix metalloprotease (MMP) target site, for site directed degradation of the polymer, XNA is a nucleic acid or nucleic acid analog, preferably a peptide nucleic acid (PNA) sequence. ### Claim 130 (depends on 129) Polymer, especially polymer as building-block for hydrogel formation, of claim 129, characterized in, that m is zero and no moiety Y is comprised in the polymer. ### Claim 131 (depends on 123, 124, 125, 126, 127, 128, 129, 130) Polymer especially polymer as building-block for hydrogel formation, according to any of the claims 123 to 130, characterized in, that it is a polymer which comprises an EDTS-moiety, preferably a MMP-moiety. ### Claim 132 (depends on 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131) Polymer, especially polymer as building-block for hydrogel formation, according to any of the claims 119 to 131, characterized in, that it comprises at least two different moieties R. ### Claim 133 (depends on 119, 120, 121) Polymer, especially polymer as building-block for hydrogel formation, according to any of the claims 119 to 121, characterized in, that p is an integer of 3 to 10, preferably 3 to 10, preferably 3 to 8, most preferred 3 to 6. ### Claim 134 Polymer, especially polymer as building-block for hydrogel formation, of formula (P2) wherein T1 is a terminating moiety, which contains a residue -XDTS-XNA1, T2 is a terminating moiety, which contains a residue -XDTS-XNA2, XDTS is independently selected from a direct bond or an EDTS-moiety, wherein EDTS is an enzyme degradable target site, preferably a matrix metalloprotease (MMP) target site, for site directed degradation of the polymer, XNA1 is a nucleic acid or nucleic acid analog, preferably a peptide nucleic acid (PNA) sequence, XNA2 is the same or a different nucleic acid or nucleic acid analog compared to XNA1, preferably a peptide nucleic acid (PNA) sequence, p is 1 or 2, preferably 1, X is a hydrophilic polymeric residue, preferably independently derived from monomers independently selected from oxazoline, ethylene glycol, propylene glycol, acetal lactic acid, glycolic acid, vinyl alcohol, n is an integer greater than 1, preferably from 1 to 10000. According to one embodiment, at least one X is different from oxazoline. ### Claim 135 (depends on 134) Polymer, especially polymer as building-block for hydrogel formation, according to formula (P2) of claim 134, characterized in that T1 is a terminating moiety, comprising no XNA-residue, T2 is a terminating moiety, comprising a XNA-residue and optionally an EDTS-moiety, p is an integer of 3 to 10, preferably 3 to 8, most preferred 3 to 6, X hydrophilic polymeric residue, preferably independently derived from monomers independently selected from oxazoline, ethylene glycol, propylene glycol, acetal lactic acid, glycolic acid, vinyl alcohol, EDTS is an enzyme degradable target site, preferably a matrix metalloprotease (MMP) target site, for site directed degradation of the polymer, XNA is a nucleic acid or nucleic acid analog, preferably a peptide nucleic acid (PNA) sequence, n is an integer greater than 1, preferably from 1 to 10000. ### Claim 136 Polymer, especially polymer as building-block for hydrogel formation, according to any of the preceding claims, wherein the polymer is functionalized by at least one biologically active compound, preferably, at least two different biologically active compounds, preferably by reaction of an amino group of the biologically active compound with a functional group of residue R4. ### Claim 137 Polymer, especially polymer as building-block for hydrogel formation, according to any of the preceding claims, characterized in, that the biologically active compound selected from the group consisting of peptides, proteins, CRISPR-Cas enzyme complex, apoptosis-inducing active substances, adhesion-promoting active substances, anti-inflammatory active substances, receptor agonists and receptor antagonists, growth-inhibiting active substances (and in particular from proteins of the extracellular matrix, cell surface proteins, antibodies, growth factors, sugars, lectins, carbohydrates, cytokines, DNA, RNA, siRNA), aptamers, and fragments thereof, or mixtures thereof. ### Claim 138 Polymer, especially polymer as building-block for hydrogel formation, according to any of the preceding claims, characterized in, that it comprises at least one biologically active compound selected from the group consisting of peptides, proteins, CRISPR-Cas enzyme complex, apoptosis-inducing active substances, adhesion-promoting active substances, anti-inflammatory active substances, receptor agonists and receptor antagonists, growth-inhibiting active substances (and in particular from proteins of the extracellular matrix, cell surface proteins, antibodies, growth factors, sugars, lectins, carbohydrates, cytokines, DNA, RNA, siRNA), aptamers, and fragments thereof, or mixtures thereof. ### Claim 139 Polymer, especially polymer as building-block for hydrogel formation, according to any of the preceding claims, characterized in, that it comprises at least one biologically active compound selected from a Peptide nucleic acid (PNA) and/or a locked nucleic acid (LNA), preferably wherein the PNA-moiety independently comprise a structure of formula (VI) wherein x is an integer greater than 1, Base is independently a moiety comprising at least one nucleobase (preferably selected from adenin, cytosin, guanine, thymine, 2,6-diaminopurine, analogs of thymine and cytosine, hypoxanthine, derivatives thereof functionalized with a fluorescent dye (preferably thiazole orange)), Rα and Rβ are independently selected from hydrogen atom, any residue bound to the alpha-carbon atom of any of the proteinogenic amino acid, Rγ is a hydrogen atom, a moiety with at least one ionic residue. ### Claim 140 Polymer, especially polymer as building-block for hydrogel formation, according to any of the preceding claims, characterized in, that it comprises at least one biologically active compound, selected from a Peptide nucleic acid (PNA) comprising a matrix metalloprotease target site for the site directed degradation (MMP). ### Claim 141 (depends on 139, 140) Polymer, especially polymer as building-block for hydrogel formation, according to claim 139 or claim 140, characterized in, that is comprises at least one additional biologically active compound, selected from the group consisting of peptides, proteins, CRISPR-Cas enzyme complex, apoptosis-inducing active substances, adhesion-promoting active substances, anti-inflammatory active substances, receptor agonists and receptor antagonists, growth-inhibiting active substances (and in particular from proteins of the extracellular matrix, cell surface proteins, antibodies, growth factors, sugars, lectins, carbohydrates, cytokines, DNA, RNA, siRNA), aptamers, and fragments thereof, or mixtures thereof. ### Claim 142 Polymer, especially polymer as building-block for hydrogel formation, according to any one of the preceding claims, wherein the polymer has a linear structure (preferably a graft polymer, grafted with at least one residue R4) or a dendritic structure (preferably a linear structure or a star shaped structure). ### Claim 143 Polymer, especially polymer as building-block for hydrogel formation, according to any one of the preceding claims, wherein the polymer is random polymer, a block-copolymer or a dendrimer. ### Claim 144 Polymer, especially polymer as building-block for hydrogel formation, according to any one of the preceding claims, wherein the polymer has a star-shaped structure comprising at least three arms. ### Claim 145 Polymer, especially polymer as building-block for hydrogel formation, according to any of the preceding claims, wherein said functional group for crosslinking is selected from amine, N-hydroxysuccinimide, sulfo-N-hydroxysuccinimide, isothiocyanate, maleimide, thiol, azide, alkyne, alkene, hydrazide, aminoxy, aldehyde, carboxyl, carboxylate, hydroxyl, acrylate, vinyl ether, epoxide (preferably from amine, maleimide, alkyne, alkene, azide, carboxyl, carboxylate, methacrylate, acrylate, thiol). ### Claim 146 Polymer, especially polymer as building-block for hydrogel formation, according to any of the preceding claims, wherein said functional group for binding a biologically active compound is independently selected from amine, N-hydroxysuccinimide, sulfo-N-hydroxysuccinimide, alkyne, alkene, hydrazide, epoxide, glycidyl, carboxyphenyl, methoxycarbonyl, carboxyl, carboxylate, isothiocyanate, maleimide, aminoxy, hydroxyl, vinyl ether (preferably from amine, N-hydroxysuccinimide, sulfo-N-hydroxysuccinimide, hydrazide, epoxide, glycidyl, phenyl acrylate, methoxycarbonyl, carboxyl, carboxylate). ### Claim 147 Polymer, especially polymer as building-block for hydrogel formation, according to any of the preceding claims, wherein the polymer is prepared by at least one polymerization step, selected from living cationic ring-opening polymerization (CROP), spontaneous zwitterionic copolymerization (SZWIP) or a combination of both. ### Claim 148 (depends on 147) Polymer, especially polymer as building-block for hydrogel formation, according to claim 147, characterized in, that the polymerization, preferably the living cationic ring-opening polymerization, is initiated by an initiator with an electrophilic character. ### Claim 149 (depends on 147, 148) Polymer, especially polymer as building-block for hydrogel formation, according to claim 147 or claim 148, characterized in, that the initiator is selected from triethylene glycol di (p)-toluenesulfonate, pentaerythritol tetrabromide, pentaerythritol tetrakis(benzenesulfonate) or p-toluenesulfonyl chloride modified N,N,N′,N′-Tetrakis(2-hydroxyethyl)ethylenediamine. ### Claim 150 (depends on 147, 148, 149) Polymer, especially polymer as building-block for hydrogel formation, according to any of claims 147 to 149, characterized in, that the polymerization, preferably the living cationic ring-opening polymerization, is terminated by addition of a terminating molecule selected from nucleophiles, amines, azides or acids (preferably carboxylic acids). ### Claim 151 (depends on 147, 148, 149, 150) Polymer, especially polymer as building-block for hydrogel formation, according to any of any of claims 147 to 150, characterized in, that the polymerization, preferably the living cationic ring-opening polymerization, is terminated by addition of a terminating molecule selected from peptide nucleic acid (PNA), preferably peptide nucleic acid (PNA) with unprotected carboxylic acid group at the C-terminus and protected amino group at the N-terminus or peptide nucleic acid (PNA) with unprotected amino group at the N-terminus and protected carboxylic acid group at the C-terminus). ### Claim 152 (depends on 147, 148, 149, 150, 151) Polymer, especially polymer as building-block for hydrogel formation, according to any of any of claims 147 to 151, characterized in, that the polymerization, preferably the spontaneous zwitterionic copolymerization, is terminated by addition of a terminating molecule selected from electrophiles, preferably selected from α,β-unsaturated carboxylic acids, α,β-unsaturated carboxylic acidamides, mixtures thereof, most preferred from acrylic acid, methacrylic acid, acryl amide, methacryl amide, functionalized with at least one residue R4 as defined in any of the preceding claims respectively (most preferred functionalized with -MMP-PNA respectively). ### Claim 153 (depends on 101, 108, 109, 110, 145, 146, 147, 148, 149, 150, 151, 152) Polymer, especially polymer as building-block for hydrogel formation, according to any of any of claims 147 to 152, characterized in, that said initiator and/or said terminating molecule incorporates a moiety R4 as defined in any of claims 101, 108 to 110, 145 and 146. ### Claim 154 (depends on 147, 148, 149, 150, 151, 153) Polymer, especially polymer as building block for hydrogel formation, according to any of the claims 147 to 151 and 153, characterized in, that the polymerization, preferably the spontaneous zwitterionic copolymerization, is terminated by addition of a terminating molecule selected from selected from α,β-unsaturated carboxylic acids, α,β-unsaturated carboxylic acidamides, mixtures thereof (most preferred from acrylic acid, methacrylic acid, acryl amide, methacryl amide) followed after optional workup by a coupling of a residue comprising PNA and a thiol functionality. ### Claim 155 (depends on 147, 148, 149, 150, 151, 152, 153, 154) Polymer, especially polymer as building block for hydrogel formation, according to any of the claims 147 to 154, characterized in, that a residue comprising PNA and a thiol functionality is coupled to a maleimide as a functional group of residue R4.