Systems, methods and hydrogels for cell culture and analysis
AssigneeEvorion Biotechnologies GmbH
InventorsHans KLEINE-BRÜGGENEY, Robert WEINGARTEN, Sebastian BÜHREN
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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.
Claims155
A hydrogel which comprises cross-linked hydrogel precursor molecules of the same type or of different types.
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.
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.
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.
The hydrogel according to any of the preceding claims, wherein the hydrogel comprises (i) linear and (ii) multiarm 2-oxazoline-based polymers.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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).
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).
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.
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.
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).
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).
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.
Dependencies are inferred from the claim text.