Thermoreversible polymers and methods of use thereof

Abstract

The present disclosure provides thermoreversible polymers, hydrogel compositions comprising the thermoreversible polymers, and methods of making and using the thermoreversible polymers. In some cases, the thermoreversible polymers comprise N-isopropylacrylamide (NIPAM) comonomers, lower alkylamine comonomers, and polyethylene glycol (PEG) comonomers. TIFF2024521148000123.tif99130

Description

[Technical field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 192,311, filed May 24, 2021, which is incorporated by reference in its entirety. [Background technology]

[0002] Introduction Patients suffering from a wide range of disorders involving tissue degeneration, such as Parkinson's disease, myocardial infarction (heart attack), or liver failure, may benefit from transplanting new healthy cells or artificial tissue to replace damaged or diseased tissue, a process known as cell replacement therapy. Stem cells have the unique ability to replicate indefinitely in an immature state and differentiate into various cell types in the body. Thus, stem cells can be utilized as a cell source for such cell replacement and tissue engineering therapies. Thus, systems and methods for scalable stem cell proliferation and differentiation are of interest. Summary of the Invention

[0003] overview The present disclosure provides thermoreversible polymers, hydrogel compositions comprising the thermoreversible polymers, and methods of making and using the thermoreversible polymers. [Brief description of the drawings]

[0004] [Figure 1-1] FIG. 1 is a schematic diagram of the synthesis of thermoreversible polymers of the present disclosure. [Figure 1-2] FIG. 1 is a schematic diagram of the synthesis of thermoreversible polymers of the present disclosure. [Diagram 2] 2A-B show the structural features of the thermoreversible polymer of the present disclosure. [Figure 3-1] 3A-3C show the mechanical properties of the thermoreversible polymers of the present disclosure. [Figure 3-2] 3D-3G show the mechanical properties of the thermoreversible polymers of the present disclosure. [Figure 4] 4A-4D demonstrate the reproducibility and scalability of the thermoreversible polymers of the present disclosure. [Figure 5A-1] 1 illustrates functionalization of thermoreversible polymers of the present disclosure. [Figure 5A-2] 1 illustrates functionalization of thermoreversible polymers of the present disclosure. [Figure 5B-1] 1 illustrates functionalization of thermoreversible polymers of the present disclosure. [Figure 5B-2] 1 illustrates functionalization of thermoreversible polymers of the present disclosure. [Figure 6] 6A-6E show the viability and proliferation of human pluripotent stem cells (hPSCs) in biomaterials comprising a thermoreversible polymer of the present disclosure. [Figure 7] 7A-7I show differentiation of hPSCs in hydrogels or Matrigel of the present disclosure. [Figure 8-1] FIG. 1 is a schematic diagram of the synthesis of a thermoreversible polymer (graft copolymer; GCP) of the present disclosure using butyl methacrylate (BMA). [Figure 8-2] FIG. 1 is a schematic diagram of the synthesis of a thermoreversible polymer (graft copolymer; GCP) of the present disclosure using butyl methacrylate (BMA). [Figure 9A] Schematic representation of functionalization of GCPs. [Figure 9B] Schematic representation of functionalization of GCPs. [Figure 9C] Schematic representation of functionalization of GCPs. [Figure 10A] 4 shows the rheological analysis of functionalized GCPs. [Figure 10B] 4 shows the rheological analysis of functionalized GCPs. [Figure 10C] 4 shows the rheological analysis of functionalized GCPs. [Figure 10D] 4 shows the rheological analysis of functionalized GCPs. [Figure 11A] Schematic representation of functionalization of GCPs. [Figure 11B] Schematic representation of functionalization of GCPs. [Figure 11C] Schematic representation of functionalization of GCPs. [Figure 11D] Schematic representation of functionalization of GCPs. [Figure 12] Schematic representation of GCP-RAFT synthesis. [Figure 13] 13A-13C show data showing the mechanical properties of GCP-BMA. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0005] definition The following terms have the following meanings unless otherwise indicated: Any undefined terms are intended to have their art-recognized meanings.

[0006] The term "cell culture" or "culturing of cells" refers to the maintenance, transportation, isolation, cultivation, propagation, passage, or differentiation of cells or tissues. Cells may be present in any arrangement or organization, such as individual cells, monolayers, cell clusters, or spheroids.

[0007] As used herein, the term "linker" or "bond" refers to a linking moiety that connects two groups and has a backbone of 100 atoms or less in length. The linker or bond may be a covalent bond connecting two groups or a covalent bond connecting a chain of 1-100 atoms in length, e.g., 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, or 20 carbon atoms, and the linker may be linear, branched, cyclic, or a single atom. In certain cases, optionally, 1, 2, 3, 4, or 5 or more carbon atoms of the linker backbone may be substituted with sulfur, nitrogen, or oxygen heteroatoms. The bonds between the backbone atoms may be saturated or unsaturated, and typically no more than 1, 2, or 3 unsaturated bonds are present in the linker backbone. The linker may include one or more substituents, e.g., with alkyl, aryl, or alkenyl groups. Linkers may include, but are not limited to, poly(ethylene glycol), ethers, thioethers, tertiary amines, straight or branched chain alkyls, such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), and the like. The linker backbone may include a cyclic group, such as an aryl, heterocycle, or cycloalkyl group, with two or more atoms of the cyclic group included in the backbone, such as 2, 3, or 4 atoms. The linker may be cleavable or non-cleavable.

[0008] "Alkyl" refers to a monovalent saturated aliphatic hydrocarbyl group having 1 to 10 carbon atoms, e.g., 1 to 6 carbon atoms, or 1 to 5, or 1 to 4, or 1 to 3 carbon atoms. In some cases, a "lower alkyl" is an alkyl group having 1 to 6 carbon atoms. This term includes straight-chain and branched-chain hydrocarbyl groups, such as, for example, methyl (CH3-), ethyl (CH3CH2-), n-propyl (CH3CH2CH2-), isopropyl ((CH3)2CH-), n-butyl (CH3CH2CH2CH2-), isobutyl ((CH3)2CHCH2-), sec-butyl ((CH3)(CH3CH2)CH-), t-butyl ((CH3)3C-), n-pentyl (CH3CH2CH2CH2CH2-), and neopentyl ((CH3)3CCH2-).

[0009] The term "substituted alkyl" refers to one or more carbon atoms in the alkyl chain that are optionally substituted with -O-, -N-, -S-, -S(O), n - (where n is 0-2), -NR- (where R is hydrogen or alkyl), and substituted with a heteroatom such as alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-aryl, -SO2-heteroaryl, and -NR a R bwherein R' and R" may be the same or different and are selected from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl, and heterocycle.

[0010] As used herein, the terms "chemoselective functional group" and "chemoselective tag" are used interchangeably and refer to chemoselective reactive groups that selectively react with each other to form a covalent bond. Chemoselective functional groups of interest include, but are not limited to, two thiol groups, a thiol and a maleimide or an iodoacetamide, and groups that can react with each other via click chemistry, such as an azide group and an alkyne group (e.g., a cyclooctyne group). Chemoselective functional groups of interest include, but are not limited to, thiols, alkynes, cyclooctynes, azides, phosphines, maleimides, alkoxyamines, aldehydes, and protected versions thereof, and precursors thereof. In certain embodiments, the chemoselective functional group is a thiol. In some cases, the chemoselective functional group is a strained alkyne, such as dibenzocyclooctyne (DBCO).

[0011] The term "RAFT" is used herein in its conventional sense to refer to reversible addition-fragmentation chain transfer polymerization. In some embodiments, RAFT agents (or chain transfer agents) for use in the methods and preparation of compounds described herein include, but are not limited to, dithioesters, dithiocarbamates, trithiocarbonates, and xanthates. In some cases, the RAFT agent is In some cases, the RAFT agent is a dithiobenzoate having the structure: In some cases, the RAFT agent is a trithiocarbonate having the structure: It is a dithiocarbamate having the structure TIFF2024521148000004.tif15128.

[0012] As used herein, lower critical solution temperature (LCST) or lower solution temperature refers to the critical temperature below which the components of a mixture are miscible in all compositions. The term "lower" refers to the lower limit of the temperature interval above which the LCST exhibits partial miscibility, i.e., miscibility at only certain compositions.

[0013] Before the present invention is further described, it is to be understood that the invention is not limited to particular embodiments described, as such may, of course, be varied. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0014] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, provided that any particular limit in the stated range is excluded. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are described herein. All publications mentioned herein are incorporated by reference in connection with the publication being cited to disclose and describe the methods and / or materials.

[0016] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to a "thermoreversible polymer" includes a plurality of such polymers, a reference to a "hydrogel composition" includes a reference to one or more hydrogel compositions and equivalents thereof known to those skilled in the art, and so forth. It should be further noted that the claims may be drafted to exclude any element. Thus, this statement is intended to serve as a predicate basis for using exclusive language, such as "solely," "only," and the like, in connection with the recitation of claim elements or the use of "negative" limitations.

[0017] It is understood that certain features of the invention, which are described in the context of separate embodiments for clarity, may also be provided in combination in a single embodiment. Conversely, various features of the invention that are described in the context of a single embodiment for brevity may also be provided individually or in any suitable subcombination. All combinations of the embodiments according to the invention are specifically embraced by the invention and are disclosed herein as if each and every combination were individually and explicitly disclosed herein. Moreover, all subcombinations of the various embodiments and elements thereof are specifically embraced by the invention and are disclosed herein as if each and every such subcombination were individually and explicitly disclosed herein.

[0018] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publications by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0019] Detailed Description The present disclosure provides thermoreversible polymers, hydrogel compositions comprising the thermoreversible polymers, and methods of making and using the thermoreversible polymers.

[0020] Thermoreversible Polymers Aspects of the present disclosure include thermoreversible polymers (also referred to as "thermosensitive polymers" or "thermoresponsive polymers"). As used herein, the term "thermoreversible" is used to refer to a polymeric material that exhibits a dramatic change in its physical properties in response to a change in temperature. Thermoreversible polymers belong to a class of stimuli-responsive materials. In some cases, thermoreversible polymers are distinguished from temperature-sensitive (e.g., thermosensitive) materials whose physical properties may change continuously in response to environmental conditions. Thermoresponsive polymers may exhibit a miscibility gap in their temperature composition diagram. Depending on whether the miscibility gap is observed at high or low temperatures, there is an upper or lower critical solution temperature (abbreviated as UCST or LCST, respectively). For example, at temperatures below the LCST, the thermoresponsive polymer is miscible with the aqueous solution in which it dissolves. At temperatures above the LCST, the thermoresponsive polymer forms a solid, semi-solid, or gel with a three-dimensional (3D) structure.

[0021] In some cases, the thermoreversible polymer comprises an N-isopropylacrylamide (NIPAM) comonomer, a lower alkylamine comonomer, and a poly(ethylene glycol) (PEG) comonomer, and the terminal PEG monomer is substituted with an alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl. In certain embodiments, the lower alkylamine comonomer comprises n-butyl, isobutyl, tert-butyl, n-propyl, pentyl, isopropyl, or isopentyl, and the terminal PEG monomer is substituted with an alkoxy group. In some cases, the alkoxy group is a C1-C6 alkoxy selected from the group consisting of methoxy, ethoxy, n-propoxy, isoproxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, and isopentoxy.

[0022] In embodiments of the present disclosure, the thermoreversible polymer (e.g., as described in more detail below) may have a variety of molecular weights, e.g., from 5 kDa to 750 kDa, e.g., from 10 kDa to 500 kDa, e.g., from 15 kDa to 450 kDa, e.g., from 20 kDa to 400 kDa, e.g., from 25 kDa to 350 kDa, e.g., from 30 kDa to 300 kDa, e.g., from 35 kDa to 250 kDa, and e.g., from 40 kDa to 200 kDa. In certain embodiments, the thermoreversible polymer of interest has a molecular weight of from 10 kDa to 500 kDa.

[0023] In some cases, the thermoreversible polymer has Formula (I): TIFF2024521148000005.tif40128 polymer, During the ceremony, a, b, and c are mole fractions of comonomers, each of a, b, and c being greater than zero; PEG n is a polyethylene glycol polymer, and n is an integer from 1 to 2500; X is independently selected from C, O, and NH. In some cases, X is O. In some cases, X is NH.

[0024] R 1 is alkyl or substituted alkyl; R 2 is alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl; and G 1 and G 2 are each independently selected from a polymer segment, an end group, a linker, and an attached modifier. 1 and G 2 are each independently a chain transfer agent. For example, the chain transfer agent may be a dithioester, a dithiocarbamate, a trithiocarbonate, or a xanthate. In certain embodiments, G 1 is a carboxyl group. In a particular example, G 1 teeth, TIFF2024521148000006.tif13128, where: TIFF2024521148000007.tif1128 is G 1 and the polymer. In certain embodiments, G 2 teeth, TIFF2024521148000008.tif12128, where: TIFF2024521148000009.tif2128 is G 2 and the polymer.

[0025] In some cases, n is an integer from 1 to 25, 1 to 50, 1 to 100, 25 to 100, 50 to 100, 1 to 150, 25 to 150, 50 to 150, 100 to 150, 100 to 125, 100 to 150, 150 to 200, 200 to 250, 250 to 500, 500 to 1000, 1000 to 1500, 1500 to 2000, or 2000 to 2500. In some embodiments, the thermoreversible polymer has a variety of molecular weights, such as, for example, 5 kDa to 750 kDa, for example, 10 kDa to 500 kDa, for example, 15 kDa to 450 kDa, for example, 20 kDa to 400 kDa, for example, 25 kDa to 350 kDa, for example, 30 kDa to 300 kDa, for example, 35 kDa to 250 kDa, and for example, 40 kDa to 200 kDa. In certain embodiments, the thermoreversible polymer has a molecular weight of 10 kDa to 500 kDa.

[0026] In some embodiments, R 1 is a C1-C6 alkyl. In some examples, R1 is an alkyl group selected from the group consisting of n-butyl, isobutyl, tert-butyl, n-propyl, pentyl, isopropyl, and isopentyl. In particular examples, R 1 is n-butyl.

[0027] In some embodiments, R 2 is an alkoxy group. In some examples, R 2 is a C1-C6 alkoxy group selected from the group consisting of methoxy, ethoxy, n-propoxy, isoproxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, and isopentoxy. 2 is methoxy.

[0028] In a specific example, a>0.8, 0.2>b>0, and 0.1>c>0.

[0029] In certain embodiments, the thermoreversible polymer has formula (II): TIFF2024521148000010.tif42128 polymer, During the ceremony, n is 1 to 2500, and G 1 and G 2 are each independently selected from a polymer segment, an end group, a linker, and an attached modifier. 1 and G 2 are each independently a chain transfer agent. For example, the chain transfer agent may be a dithioester, a dithiocarbamate, a trithiocarbonate, or a xanthate. In certain embodiments, G 1 is a carboxyl group. In a particular example, G 1 teeth, TIFF2024521148000011.tif13128, where: TIFF2024521148000012.tif2128 is G 1 and the polymer. In certain embodiments, G 2 teeth, TIFF2024521148000013.tif12128, where: TIFF2024521148000014.tif2128 is G 2 and the polymer.

[0030] In certain embodiments, PEG or PEG n has a molecular weight of 2 kDa to 100 kDa.

[0031] In some cases, n is an integer from 1 to 25, 1 to 50, 1 to 100, 25 to 100, 50 to 100, 1 to 150, 25 to 150, 50 to 150, 100 to 150, 100 to 125, 100 to 150, 150 to 200, 200 to 250, 250 to 500, 500 to 1000, 1000 to 1500, 1500 to 2000, or 2000 to 2500. In some embodiments, the thermoreversible polymer has a variety of molecular weights, such as, for example, 5 kDa to 750 kDa, for example, 10 kDa to 500 kDa, for example, 15 kDa to 450 kDa, for example, 20 kDa to 400 kDa, for example, 25 kDa to 350 kDa, for example, 30 kDa to 300 kDa, for example, 35 kDa to 250 kDa, and for example, 40 kDa to 200 kDa. In certain embodiments, the thermoreversible polymer has a molecular weight of 10 kDa to 500 kDa.

[0032] In some embodiments, the thermoreversible polymer has formula (III): TIFF2024521148000015.tif40128 polymer, During the ceremony, a, b, c, and d are mole fractions of comonomers, each of a, b, c, and d being greater than zero; PEG n is a polyethylene glycol polymer, and n is an integer from 1 to 2500; R 1 is alkyl or substituted alkyl; R 2 is alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl; L is a linker, Z 2 is a modifier or chemoselective functional group, and G 1 and G 2are each independently selected from a polymer segment, an end group, a linker, and an attached modifier. 1 and G 2 are each independently a chain transfer agent. For example, the chain transfer agent may be a dithioester, a dithiocarbamate, a trithiocarbonate, or a xanthate. In certain embodiments, G 1 is a carboxyl group. In a particular example, G 1 teeth, TIFF2024521148000016.tif13128, where: TIFF2024521148000017.tif2128 is G 1 and the polymer. In certain embodiments, G 2 teeth, TIFF2024521148000018.tif12128, where: TIFF2024521148000019.tif1128 is G 2 and the polymer.

[0033] In some cases, n is an integer from 1 to 25, 1 to 50, 1 to 100, 25 to 100, 50 to 100, 1 to 150, 25 to 150, 50 to 150, 100 to 150, 100 to 125, 100 to 150, 150 to 200, 200 to 250, 250 to 500, 500 to 1000, 1000 to 1500, 1500 to 2000, or 2000 to 2500. In some embodiments, the thermoreversible polymer has a variety of molecular weights, such as, for example, 5 kDa to 750 kDa, for example, 10 kDa to 500 kDa, for example, 15 kDa to 450 kDa, for example, 20 kDa to 400 kDa, for example, 25 kDa to 350 kDa, for example, 30 kDa to 300 kDa, for example, 35 kDa to 250 kDa, and for example, 40 kDa to 200 kDa. In certain embodiments, the thermoreversible polymer has a molecular weight of 10 kDa to 500 kDa.

[0034] In some embodiments, R 1is a C1-C6 alkyl. In some examples, R1 is an alkyl group selected from the group consisting of n-butyl, isobutyl, tert-butyl, n-propyl, pentyl, isopropyl, and isopentyl. In particular examples, R 1 is n-butyl.

[0035] In some embodiments, R 2 is an alkoxy group. In some examples, R 2 is a C1-C6 alkoxy group selected from the group consisting of methoxy, ethoxy, n-propoxy, isoproxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, and isopentoxy. 2 is methoxy.

[0036] In a specific example, a>0.8, 0.2>b>0, 0.1>c>0, and 0.1>d>0.

[0037] In some embodiments, Z 2 is a chemoselective functional group selected from thiols, alkynes, cyclooctynes, azides, phosphines, maleimides, alkoxyamines, aldehydes, and protected forms or precursors thereof.

[0038] In some cases, Z 2 Z includes a group such as a methacrylate. As shown in FIG. 5B, such a group can be used to attach a thiol-containing molecule. For example, the thiol-containing molecule can be a protein, peptide, heparin, etc. that contains or has been modified to contain a free thiol group. In some cases, Z 2 contains a thiol. Using standard chemistry, the free thiol can be used as an attachment point for a variety of molecules, as shown in FIG. 5B. 2 contains a strained alkyne. As shown in FIG. 5B, the strained alkyne can be used to attach a variety of molecules using well-known reactions.

[0039] In some instances, Z 2 is a modifier selected from heparin, hyaluronic acid, a specific binding member, a peptide, a fibroblast growth factor (FGF), a nucleic acid, gelatin, fibronectin, collagen, laminin, basic fibroblast growth factor (bFGF; also known as fibroblast growth factor 2 (FGF2)), FGF7, FGF8, FGF10, epidermal growth factor (EGF), insulin, progesterone, glucose, stromal cell-derived factor-1 (SDF-1), thymosin beta4, sonic hedgehog (SHH), noggin, activin, transforming growth factor beta (TGF-β) (TGFβ3), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), neurotrophic factor 3 (NT3), nerve growth factor (NGF), platelet-derived growth factor (PDGF), interleukins (e.g., IL-2 or IL-16), and insulin-like growth factor 1 (IGF-1).

[0040] In some instances, G 1 , G 2 , and Z 2 is an independently selected modifier selected from heparin, hyaluronic acid, a member of a specific binding pair, a polypeptide, and a nucleic acid. 1 , G 2 , and Z 2 One or more of are independently selected from a modifier selected from gelatin, fibronectin, collagen, or laminin.

[0041] In some instances, G 1 , G 2 , and Z 2One or more of the polypeptides are selected from chemokines, peptide hormones, or growth factors. In certain examples, the polypeptide is fibroblast growth factor, epidermal growth factor, hepatocyte growth factor, insulin, stromal cell-derived factor-1, thymosin beta4, sonic hedgehog, noggin, activin, transforming growth factor, bone morphogenetic protein, brain-derived neurotrophic factor, glial cell-derived neurotrophic factor, neurotrophin-3, platelet-derived growth factor, FGF-2, FGF-8, keratinocyte growth factor, or insulin-like growth factor. In some examples, the polypeptide is selected from hepatocyte growth factor, bone morphogenetic protein, FGF-2, FGF-8, and keratinocyte growth factor.

[0042] In some embodiments, the thermoreversible polymer has formula (IV): TIFF2024521148000020.tif47128 polymer, During the ceremony, a, b, c, and d are mole fractions of comonomers, each of a, b, c, and d being greater than zero; PEG n is a polyethylene glycol polymer, and n is an integer from 1 to 2500; R 1 is alkyl or substituted alkyl; R 2 is alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl; G 1 and G 2 are each independently selected from a polymer segment, an end group, a linker, and an attached modifier. 1 and G 2are each independently a chain transfer agent. For example, the chain transfer agent may be a dithioester, a dithiocarbamate, a trithiocarbonate, or a xanthate. In certain embodiments, G 1 is a carboxyl group. In a particular example, G 1 teeth, TIFF2024521148000021.tif13128, where: TIFF2024521148000022.tif1128 is G 1 and the polymer. In certain embodiments, G 2 teeth, TIFF2024521148000023.tif12128, where: TIFF2024521148000024.tif2128 is G 2 and the polymer.

[0043] In some cases, n is an integer from 1 to 25, 1 to 50, 1 to 100, 25 to 100, 50 to 100, 1 to 150, 25 to 150, 50 to 150, 100 to 150, 100 to 125, 100 to 150, 150 to 200, 200 to 250, 250 to 500, 500 to 1000, 1000 to 1500, 1500 to 2000, or 2000 to 2500. In some embodiments, the thermoreversible polymer has a variety of molecular weights, such as, for example, 5 kDa to 750 kDa, for example, 10 kDa to 500 kDa, for example, 15 kDa to 450 kDa, for example, 20 kDa to 400 kDa, for example, 25 kDa to 350 kDa, for example, 30 kDa to 300 kDa, for example, 35 kDa to 250 kDa, and for example, 40 kDa to 200 kDa. In certain embodiments, the thermoreversible polymer has a molecular weight of 10 kDa to 500 kDa.

[0044] In some embodiments, R 1 is a C1-C6 alkyl. In some cases, R1 is an alkyl group selected from the group consisting of n-butyl, isobutyl, tert-butyl, n-propyl, pentyl, isopropyl, and isopentyl. In certain cases, R 1is n-butyl.

[0045] In some embodiments, R 2 is an alkoxy group. In some examples, R 2 is a C1-C6 alkoxy group selected from the group consisting of methoxy, ethoxy, n-propoxy, isoproxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, and isopentoxy. 2 is methoxy.

[0046] In a specific example, a>0.8, 0.2>b>0, 0.1>c>0, and 0.1>d>0.

[0047] In some embodiments, the thermoreversible polymer has formula (V): TIFF2024521148000025.tif59128 polymer, During the ceremony, a, b, c, and d are mole fractions of comonomers, each of a, b, c, and d being greater than zero; PEG n is a polyethylene glycol polymer, and n is an integer from 1 to 2500; R 1 is alkyl or substituted alkyl; R 2 is alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl; G 1 and G 2 are each independently selected from a polymer segment, an end group, a linker, and an attached modifier. 1 and G 2are each independently a chain transfer agent. For example, the chain transfer agent may be a dithioester, a dithiocarbamate, a trithiocarbonate, or a xanthate. In certain embodiments, G 1 is a carboxyl group. In a particular example, G 1 teeth, TIFF2024521148000026.tif13128, where: TIFF2024521148000027.tif2128 is G 1 and the polymer. In certain embodiments, G 2 teeth, TIFF2024521148000028.tif12128, where: TIFF2024521148000029.tif1128 is G 2 and the polymer.

[0048] In some cases, n is an integer from 1 to 25, 1 to 50, 1 to 100, 25 to 100, 50 to 100, 1 to 150, 25 to 150, 50 to 150, 100 to 150, 100 to 125, 100 to 150, 150 to 200, 200 to 250, 250 to 500, 500 to 1000, 1000 to 1500, 1500 to 2000, or 2000 to 2500. In some embodiments, the thermoreversible polymer has a variety of molecular weights, such as, for example, 5 kDa to 750 kDa, for example, 10 kDa to 500 kDa, for example, 15 kDa to 450 kDa, for example, 20 kDa to 400 kDa, for example, 25 kDa to 350 kDa, for example, 30 kDa to 300 kDa, for example, 35 kDa to 250 kDa, and for example, 40 kDa to 200 kDa. In certain embodiments, the thermoreversible polymer has a molecular weight of 10 kDa to 500 kDa.

[0049] In some embodiments, R 1 is a C1-C6 alkyl. In some examples, R1 is an alkyl group selected from the group consisting of n-butyl, isobutyl, tert-butyl, n-propyl, pentyl, isopropyl, and isopentyl. In particular examples, R 1 is n-butyl.

[0050] In some embodiments, R 2 is an alkoxy group. In some examples, R 2 is a C1-C6 alkoxy group selected from the group consisting of methoxy, ethoxy, n-propoxy, isoproxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, and isopentoxy. 2 is methoxy.

[0051] In a specific example, a>0.8, 0.2>b>0, 0.1>c>0, and 0.1>d>0.

[0052] In some embodiments, the thermoreversible polymer has formula (VI): TIFF2024521148000030.tif50128 polymer, During the ceremony, a, b, c, and d are mole fractions of comonomers, each of a, b, c, and d being greater than zero; PEG n is a polyethylene glycol polymer, and n is an integer from 1 to 2500; R 1 is alkyl or substituted alkyl; R 2 is alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl; G 1 and G 2 are each independently selected from a polymer segment, an end group, a linker, and an attached modifier. 1 and G 2are each independently a chain transfer agent. For example, the chain transfer agent may be a dithioester, a dithiocarbamate, a trithiocarbonate, or a xanthate. In certain embodiments, G 1 is a carboxyl group. In a particular example, G 1 teeth, TIFF2024521148000031.tif13128, where: TIFF2024521148000032.tif2128 is G 1 and the polymer. In certain embodiments, G 2 teeth, TIFF2024521148000033.tif12128, where: TIFF2024521148000034.tif1128 is G 2 and the polymer.

[0053] In some cases, n is an integer from 1 to 25, 1 to 50, 1 to 100, 25 to 100, 50 to 100, 1 to 150, 25 to 150, 50 to 150, 100 to 150, 100 to 125, 100 to 150, 150 to 200, 200 to 250, 250 to 500, 500 to 1000, 1000 to 1500, 1500 to 2000, or 2000 to 2500. In some embodiments, the thermoreversible polymer has a variety of molecular weights, such as, for example, 5 kDa to 750 kDa, for example, 10 kDa to 500 kDa, for example, 15 kDa to 450 kDa, for example, 20 kDa to 400 kDa, for example, 25 kDa to 350 kDa, for example, 30 kDa to 300 kDa, for example, 35 kDa to 250 kDa, and for example, 40 kDa to 200 kDa. In certain embodiments, the thermoreversible polymer has a molecular weight of 10 kDa to 500 kDa.

[0054] In some embodiments, R 1 is a C1-C6 alkyl. In some examples, R1 is an alkyl group selected from the group consisting of n-butyl, isobutyl, tert-butyl, n-propyl, pentyl, isopropyl, and isopentyl. In certain cases, R 1 is n-butyl.

[0055] In some embodiments, R 2 is an alkoxy group. In some examples, R 2 is a C1-C6 alkoxy group selected from the group consisting of methoxy, ethoxy, n-propoxy, isoproxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, and isopentoxy. 2 is methoxy.

[0056] In a specific example, a>0.8, 0.2>b>0, 0.1>c>0, and 0.1>d>0.

[0057] In some embodiments, the thermoreversible polymer has formula (VII): TIFF2024521148000035.tif41128 polymer, During the ceremony, a, b, c, and d are mole fractions of comonomers, each of a, b, c, and d being greater than zero; PEG n is a polyethylene glycol polymer, and n is an integer from 1 to 2500; R 1 is alkyl or substituted alkyl; R 2 is alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl; G 1 and G 2 are each independently selected from a polymer segment, an end group, a linker, and an attached modifier. 1 and G 2are each independently a chain transfer agent. For example, the chain transfer agent may be a dithioester, a dithiocarbamate, a trithiocarbonate, or a xanthate. In certain embodiments, G 1 is a carboxyl group. In a particular example, G 1 teeth, TIFF2024521148000036.tif13128, where: TIFF2024521148000037.tif1128 is G 1 and the polymer. In certain embodiments, G 2 teeth, TIFF2024521148000038.tif12128, where: TIFF2024521148000039.tif1128 is G 2 and the polymer.

[0058] In some cases, n is an integer from 1 to 25, 1 to 50, 1 to 100, 25 to 100, 50 to 100, 1 to 150, 25 to 150, 50 to 150, 100 to 150, 100 to 125, 100 to 150, 150 to 200, 200 to 250, 250 to 500, 500 to 1000, 1000 to 1500, 1500 to 2000, or 2000 to 2500. In some embodiments, the thermoreversible polymer has a variety of molecular weights, such as, for example, 5 kDa to 750 kDa, for example, 10 kDa to 500 kDa, for example, 15 kDa to 450 kDa, for example, 20 kDa to 400 kDa, for example, 25 kDa to 350 kDa, for example, 30 kDa to 300 kDa, for example, 35 kDa to 250 kDa, and for example, 40 kDa to 200 kDa. In certain embodiments, the thermoreversible polymer has a molecular weight of 10 kDa to 500 kDa.

[0059] In some embodiments, R 1 is a C1-C6 alkyl. In some cases, R1 is an alkyl group selected from the group consisting of n-butyl, isobutyl, tert-butyl, n-propyl, pentyl, isopropyl, and isopentyl. In certain cases, R 1is n-butyl.

[0060] In some embodiments, R 2 is an alkoxy group. In some examples, R 2 is a C1-C6 alkoxy group selected from the group consisting of methoxy, ethoxy, n-propoxy, isoproxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, and isopentoxy. 2 is methoxy.

[0061] In a specific example, a>0.8, 0.2>b>0, 0.1>c>0, and 0.1>d>0.

[0062] In some embodiments, the thermoreversible polymer has formula (VIII): TIFF2024521148000040.tif40128 polymer, During the ceremony, a, b, c, and d are mole fractions of comonomers, each of a, b, c, and d being greater than zero; PEG n is a polyethylene glycol polymer, and n is an integer from 1 to 2500; R 1 is alkyl or substituted alkyl; R 2 is alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl; G 1 and G 2 are each independently selected from a polymer segment, an end group, a linker, and an attached modifier. 1 and G 2are each independently a chain transfer agent. For example, the chain transfer agent may be a dithioester, a dithiocarbamate, a trithiocarbonate, or a xanthate. In certain embodiments, G 1 is a carboxyl group. In a particular example, G 1 teeth, TIFF2024521148000041.tif13128, where: TIFF2024521148000042.tif2128 is G 1 and the polymer. In certain embodiments, G 2 teeth, TIFF2024521148000043.tif12128, where: TIFF2024521148000044.tif2128 is G 2 and the polymer.

[0063] In some cases, n is an integer from 1 to 25, 1 to 50, 1 to 100, 25 to 100, 50 to 100, 1 to 150, 25 to 150, 50 to 150, 100 to 150, 100 to 125, 100 to 150, 150 to 200, 200 to 250, 250 to 500, 500 to 1000, 1000 to 1500, 1500 to 2000, or 2000 to 2500. In some embodiments, the thermoreversible polymer has a variety of molecular weights, such as, for example, 5 kDa to 750 kDa, for example, 10 kDa to 500 kDa, for example, 15 kDa to 450 kDa, for example, 20 kDa to 400 kDa, for example, 25 kDa to 350 kDa, for example, 30 kDa to 300 kDa, for example, 35 kDa to 250 kDa, and for example, 40 kDa to 200 kDa. In certain embodiments, the thermoreversible polymer has a molecular weight of 10 kDa to 500 kDa.

[0064] In some embodiments, R 1 is a C1-C6 alkyl. In some examples, R1 is an alkyl group selected from the group consisting of n-butyl, isobutyl, tert-butyl, n-propyl, pentyl, isopropyl, and isopentyl. In particular examples, R 1 is n-butyl.

[0065] In some embodiments, R 2 is an alkoxy group. In some examples, R 2 is a C1-C6 alkoxy group selected from the group consisting of methoxy, ethoxy, n-propoxy, isoproxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, and isopentoxy. 2 is methoxy.

[0066] In a specific example, a>0.8, 0.2>b>0, 0.1>c>0, and 0.1>d>0.

[0067] In some embodiments, the thermoreversible polymer has formula (IX): TIFF2024521148000045.tif40128 polymer, During the ceremony, a, b, c, and d are mole fractions of comonomers, each of a, b, c, and d being greater than zero; PEG n is a polyethylene glycol polymer, and n is an integer from 1 to 2500; R 1 is alkyl or substituted alkyl; R 2 is alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl; G 1 and G 2 are each independently selected from a polymer segment, an end group, a linker, and an attached modifier. 1 and G 2are each independently a chain transfer agent. For example, the chain transfer agent may be a dithioester, a dithiocarbamate, a trithiocarbonate, or a xanthate. In certain embodiments, G 1 is a carboxyl group. In a particular example, G 1 teeth, TIFF2024521148000046.tif13128, where: TIFF2024521148000047.tif2128 is G 1 and the polymer. In certain embodiments, G 2 teeth, TIFF2024521148000048.tif12128, where: TIFF2024521148000049.tif1128 is G 2 and the polymer.

[0068] In some cases, n is an integer from 1 to 25, 1 to 50, 1 to 100, 25 to 100, 50 to 100, 1 to 150, 25 to 150, 50 to 150, 100 to 150, 100 to 125, 100 to 150, 150 to 200, 200 to 250, 250 to 500, 500 to 1000, 1000 to 1500, 1500 to 2000, or 2000 to 2500. In some embodiments, the thermoreversible polymer has a variety of molecular weights, such as, for example, 5 kDa to 750 kDa, for example, 10 kDa to 500 kDa, for example, 15 kDa to 450 kDa, for example, 20 kDa to 400 kDa, for example, 25 kDa to 350 kDa, for example, 30 kDa to 300 kDa, for example, 35 kDa to 250 kDa, and for example, 40 kDa to 200 kDa. In certain embodiments, the thermoreversible polymer has a molecular weight of 10 kDa to 500 kDa.

[0069] In some embodiments, R 1 is a C1-C6 alkyl. In some examples, R1 is an alkyl group selected from the group consisting of n-butyl, isobutyl, tert-butyl, n-propyl, pentyl, isopropyl, and isopentyl. In particular examples, R 1 is n-butyl.

[0070] In some embodiments, R 2 is an alkoxy group. In some examples, R 2 is a C1-C6 alkoxy group selected from the group consisting of methoxy, ethoxy, n-propoxy, isoproxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, and isopentoxy. 2 is methoxy.

[0071] In a specific example, a>0.8, 0.2>b>0, 0.1>c>0, and 0.1>d>0.

[0072] In some embodiments, the thermoreversible polymer has formula (X): TIFF2024521148000050.tif47128 polymer, During the ceremony, a, b, c, and d are mole fractions of comonomers, each of a, b, c, and d being greater than zero; PEG n is a polyethylene glycol polymer, n is an integer from 1 to 2500, R 1 is alkyl or substituted alkyl; R 2 is alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl; G 1 and G 2 are each independently selected from a polymer segment, an end group, a linker, and an attached modifier. 1 and G 2are each independently a chain transfer agent. For example, the chain transfer agent may be a dithioester, a dithiocarbamate, a trithiocarbonate, or a xanthate. In certain embodiments, G 1 is a carboxyl group. In a particular example, G 1 teeth, TIFF2024521148000051.tif13128, where: TIFF2024521148000052.tif2128 is G 1 and the polymer. In certain embodiments, G 2 teeth, TIFF2024521148000053.tif12128, where: TIFF2024521148000054.tif1128 is G 2 and the polymer.

[0073] In some examples, n is an integer between 1 and 25, 1 and 50, 1 and 100, 25 and 100, 50 and 100, 1 and 150, 25 and 150, 50 and 150, 100 and 150, 100 and 125, 100 and 150, 150 and 200 and 250, 250 and 500, 500 and 1000, 1000 and 1500, 1500 and 2000, or 2000 and 2500. In some embodiments, the thermoreversible polymer has a variety of molecular weights, such as, for example, 5 kDa to 750 kDa, for example, 10 kDa to 500 kDa, for example, 15 kDa to 450 kDa, for example, 20 kDa to 400 kDa, for example, 25 kDa to 350 kDa, for example, 30 kDa to 300 kDa, for example, 35 kDa to 250 kDa, and for example, 40 kDa to 200 kDa. In certain embodiments, the thermoreversible polymer has a molecular weight of 10 kDa to 500 kDa.

[0074] In some embodiments, R 1 is a C1-C6 alkyl. In some examples, R1 is an alkyl group selected from the group consisting of n-butyl, isobutyl, tert-butyl, n-propyl, pentyl, isopropyl, and isopentyl. In particular examples, R 1 is n-butyl.

[0075] In some embodiments, R 2 is an alkoxy group. In some examples, R 2 is a C1-C6 alkoxy group selected from the group consisting of methoxy, ethoxy, n-propoxy, isoproxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, and isopentoxy. 2 is methoxy.

[0076] In a specific example, a>0.8, 0.2>b>0, 0.1>c>0, and 0.1>d>0.

[0077] In some embodiments, the thermoreversible polymer has formula (XI): TIFF2024521148000055.tif59128 polymer, During the ceremony, a, b, c, and d are mole fractions of comonomers, each of a, b, c, and d being greater than zero; PEG n is a polyethylene glycol polymer, and n is an integer from 1 to 2500; R 1 is alkyl or substituted alkyl; R 2 is alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl; G 1 and G 2 are each independently selected from a polymer segment, an end group, a linker, and an attached modifier. 1 and G 2are each independently a chain transfer agent. For example, the chain transfer agent may be a dithioester, a dithiocarbamate, a trithiocarbonate, or a xanthate. In certain embodiments, G 1 is a carboxyl group. In a particular example, G 1 teeth, TIFF2024521148000056.tif13128, where: TIFF2024521148000057.tif2128 is G 1 and the polymer. In certain embodiments, G 2 teeth, TIFF2024521148000058.tif12128, where: TIFF2024521148000059.tif1128 is G 2 and the polymer.

[0078] In some cases, n is an integer from 1 to 25, 1 to 50, 1 to 100, 25 to 100, 50 to 100, 1 to 150, 25 to 150, 50 to 150, 100 to 150, 100 to 125, 100 to 150, 150 to 200, 200 to 250, 250 to 500, 500 to 1000, 1000 to 1500, 1500 to 2000, or 2000 to 2500. In some embodiments, the thermoreversible polymer has a variety of molecular weights, such as, for example, 5 kDa to 750 kDa, for example, 10 kDa to 500 kDa, for example, 15 kDa to 450 kDa, for example, 20 kDa to 400 kDa, for example, 25 kDa to 350 kDa, for example, 30 kDa to 300 kDa, for example, 35 kDa to 250 kDa, and for example, 40 kDa to 200 kDa. In certain embodiments, the thermoreversible polymer has a molecular weight of 10 kDa to 500 kDa.

[0079] In some embodiments, R 1 is a C1-C6 alkyl. In some examples, R1 is an alkyl group selected from the group consisting of n-butyl, isobutyl, tert-butyl, n-propyl, pentyl, isopropyl, and isopentyl. In particular examples, R 1 is n-butyl.

[0080] In some embodiments, R 2 is an alkoxy group. In some examples, R 2 is a C1-C6 alkoxy group selected from the group consisting of methoxy, ethoxy, n-propoxy, isoproxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, and isopentoxy. 2 is methoxy.

[0081] In a specific example, a>0.8, 0.2>b>0, 0.1>c>0, and 0.1>d>0.

[0082] In some embodiments, the thermoreversible polymer has formula (XII): TIFF2024521148000060.tif58128 polymer, During the ceremony, a, b, c, and d are mole fractions of comonomers, each of a, b, c, and d being greater than zero; PEG n is a polyethylene glycol polymer, and n is an integer from 1 to 2500; R 1 is alkyl or substituted alkyl; R 2 is alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl; G 1 and G 2 are each independently selected from a polymer segment, an end group, a linker, and an attached modifier. 1 and G 2are each independently a chain transfer agent. For example, the chain transfer agent may be a dithioester, a dithiocarbamate, a trithiocarbonate, or a xanthate. In certain embodiments, G 1 is a carboxyl group. In a particular example, G 1 teeth, TIFF2024521148000061.tif13128, where: TIFF2024521148000062.tif2128 is G 1 and the polymer. In certain embodiments, G 2 teeth, TIFF2024521148000063.tif12128, where: TIFF2024521148000064.tif1128 is G 2 and the polymer.

[0083] In some cases, n is an integer from 1 to 25, 1 to 50, 1 to 100, 25 to 100, 50 to 100, 1 to 150, 25 to 150, 50 to 150, 100 to 150, 100 to 125, 100 to 150, 150 to 200, 200 to 250, 250 to 500, 500 to 1000, 1000 to 1500, 1500 to 2000, or 2000 to 2500. In some embodiments, the thermoreversible polymer has a variety of molecular weights, such as, for example, 5 kDa to 750 kDa, for example, 10 kDa to 500 kDa, for example, 15 kDa to 450 kDa, for example, 20 kDa to 400 kDa, for example, 25 kDa to 350 kDa, for example, 30 kDa to 300 kDa, for example, 35 kDa to 250 kDa, and for example, 40 kDa to 200 kDa. In certain embodiments, the thermoreversible polymer has a molecular weight of 10 kDa to 500 kDa.

[0084] In some embodiments, R 1 is a C1-C6 alkyl. In some examples, R1 is an alkyl group selected from the group consisting of n-butyl, isobutyl, tert-butyl, n-propyl, pentyl, isopropyl, and isopentyl. In particular examples, R 1 is n-butyl.

[0085] In some embodiments, R 2 is an alkoxy group. In some examples, R 2 is a C1-C6 alkoxy group selected from the group consisting of methoxy, ethoxy, n-propoxy, isoproxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, and isopentoxy. 2 is methoxy.

[0086] In a specific example, a>0.8, 0.2>b>0, 0.1>c>0, and 0.1>d>0.

[0087] In some embodiments, the thermoreversible polymer has formula (XIII): TIFF2024521148000065.tif42128 polymer, During the ceremony, a, b, c, and d are mole fractions of comonomers, each of a, b, c, and d being greater than zero; PEG n is a polyethylene glycol polymer, and n is an integer from 1 to 2500; R 1 is alkyl or substituted alkyl; R 2 is alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl; G 1 and G 2 are each independently selected from a polymer segment, an end group, a linker, and an attached modifier. 1 and G 2are each independently a chain transfer agent. For example, the chain transfer agent may be a dithioester, a dithiocarbamate, a trithiocarbonate, or a xanthate. In certain embodiments, G 1 is a carboxyl group. In a particular example, G 1 teeth, TIFF2024521148000066.tif13128, where: TIFF2024521148000067.tif1128 is G 1 and the polymer. In certain embodiments, G 2 teeth, TIFF2024521148000068.tif12128, where: TIFF2024521148000069.tif2128 is G 2 and the polymer.

[0088] In some cases, n is an integer from 1 to 25, 1 to 50, 1 to 100, 25 to 100, 50 to 100, 1 to 150, 25 to 150, 50 to 150, 100 to 150, 100 to 125, 100 to 150, 150 to 200, 200 to 250, 250 to 500, 500 to 1000, 1000 to 1500, 1500 to 2000, or 2000 to 2500. In some embodiments, the thermoreversible polymer has a variety of molecular weights, such as, for example, 5 kDa to 750 kDa, for example, 10 kDa to 500 kDa, for example, 15 kDa to 450 kDa, for example, 20 kDa to 400 kDa, for example, 25 kDa to 350 kDa, for example, 30 kDa to 300 kDa, for example, 35 kDa to 250 kDa, and for example, 40 kDa to 200 kDa. In certain embodiments, the thermoreversible polymer has a molecular weight of 10 kDa to 500 kDa.

[0089] In some embodiments, R 1 is a C1-C6 alkyl. In some examples, R1 is an alkyl group selected from the group consisting of n-butyl, isobutyl, tert-butyl, n-propyl, pentyl, isopropyl, and isopentyl. In particular examples, R 1 is n-butyl.

[0090] In some embodiments, R 2 is an alkoxy group. In some examples, R 2 is a C1-C6 alkoxy group selected from the group consisting of methoxy, ethoxy, n-propoxy, isoproxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, and isopentoxy. 2 is methoxy.

[0091] In a specific example, a>0.8, 0.2>b>0, 0.1>c>0, and 0.1>d>0.

[0092] In some embodiments, the thermoreversible polymer has formula (XIV): TIFF2024521148000070.tif40128 polymer, During the ceremony, a, b, c, and d are mole fractions of comonomers, each of a, b, c, and d being greater than zero; PEG n is a polyethylene glycol polymer, and n is an integer from 1 to 2500; R 1 is alkyl or substituted alkyl; R 2 is alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl; G 1 and G 2 are each independently selected from a polymer segment, an end group, a linker, and an attached modifier. 1 and G 2are each independently a chain transfer agent. For example, the chain transfer agent may be a dithioester, a dithiocarbamate, a trithiocarbonate, or a xanthate. In certain embodiments, G 1 is a carboxyl group. In a particular example, G 1 teeth, TIFF2024521148000071.tif13128, where: TIFF2024521148000072.tif2128 is G 1 and the polymer. In certain embodiments, G 2 teeth, TIFF2024521148000073.tif12128, where: TIFF2024521148000074.tif2128 is G 2 and the polymer.

[0093] In some cases, n is an integer from 1 to 25, 1 to 50, 1 to 100, 25 to 100, 50 to 100, 1 to 150, 25 to 150, 50 to 150, 100 to 150, 100 to 125, 100 to 150, 150 to 200, 200 to 250, 250 to 500, 500 to 1000, 1000 to 1500, 1500 to 2000, or 2000 to 2500. In some embodiments, the thermoreversible polymer has a variety of molecular weights, such as, for example, 5 kDa to 750 kDa, for example, 10 kDa to 500 kDa, for example, 15 kDa to 450 kDa, for example, 20 kDa to 400 kDa, for example, 25 kDa to 350 kDa, for example, 30 kDa to 300 kDa, for example, 35 kDa to 250 kDa, and for example, 40 kDa to 200 kDa. In certain embodiments, the thermoreversible polymer has a molecular weight of 10 kDa to 500 kDa.

[0094] In some embodiments, R 1 is a C1-C6 alkyl. In some examples, R1 is an alkyl group selected from the group consisting of n-butyl, isobutyl, tert-butyl, n-propyl, pentyl, isopropyl, and isopentyl. In particular examples, R 1 is n-butyl.

[0095] In some embodiments, R 2 is an alkoxy group. In some examples, R 2 is a C1-C6 alkoxy group selected from the group consisting of methoxy, ethoxy, n-propoxy, isoproxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, and isopentoxy. 2 is methoxy.

[0096] In a specific example, a>0.8, 0.2>b>0, 0.1>c>0, and 0.1>d>0.

[0097] In some embodiments, the thermoreversible polymer has formula (XV): TIFF2024521148000075.tif40128 polymer, During the ceremony, a, b, c, and d are mole fractions of comonomers, each of a, b, c, and d being greater than zero; PEG n is a polyethylene glycol polymer, and n is an integer from 1 to 2500; R 1 is alkyl or substituted alkyl; R 2 is alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl; G 1 and G 2 are each independently selected from a polymer segment, an end group, a linker, and an attached modifier. 1 and G 2are each independently a chain transfer agent. For example, the chain transfer agent may be a dithioester, a dithiocarbamate, a trithiocarbonate, or a xanthate. In certain embodiments, G 1 is a carboxyl group. In a particular example, G 1 teeth, TIFF2024521148000076.tif13128, where: TIFF2024521148000077.tif2128 is G 1 and the polymer. In certain embodiments, G 2 teeth, TIFF2024521148000078.tif12128, where: TIFF2024521148000079.tif2128 is G 2 and the polymer.

[0098] In some cases, n is an integer from 1 to 25, 1 to 50, 1 to 100, 25 to 100, 50 to 100, 1 to 150, 25 to 150, 50 to 150, 100 to 150, 100 to 125, 100 to 150, 150 to 200, 200 to 250, 250 to 500, 500 to 1000, 1000 to 1500, 1500 to 2000, or 2000 to 2500. In some embodiments, the thermoreversible polymer has a variety of molecular weights, such as, for example, 5 kDa to 750 kDa, for example, 10 kDa to 500 kDa, for example, 15 kDa to 450 kDa, for example, 20 kDa to 400 kDa, for example, 25 kDa to 350 kDa, for example, 30 kDa to 300 kDa, for example, 35 kDa to 250 kDa, and for example, 40 kDa to 200 kDa. In certain embodiments, the thermoreversible polymer has a molecular weight of 10 kDa to 500 kDa.

[0099] In some embodiments, R 1 is a C1-C6 alkyl. In some examples, R1 is an alkyl group selected from the group consisting of n-butyl, isobutyl, tert-butyl, n-propyl, pentyl, isopropyl, and isopentyl. In particular examples, R 1 is n-butyl.

[0100] In some embodiments, R 2 is an alkoxy group. In some examples, R 2 is a C1-C6 alkoxy group selected from the group consisting of methoxy, ethoxy, n-propoxy, isoproxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, and isopentoxy. 2 is methoxy.

[0101] In a specific example, a>0.8, 0.2>b>0, 0.1>c>0, and 0.1>d>0.

[0102] Method for preparing thermoreversible polymers The present disclosure provides a method for making the thermoreversible polymer of the present disclosure. In some cases, the method includes copolymerizing N-isopropylacrylamide and N-acryloxysuccinimide to produce a first copolymer comprising an acrylamide backbone, contacting the copolymer with an alkylamine and an alkoxypolyethylene glycol amine to produce a second copolymer, and contacting the second copolymer with isopropylamine to produce a thermoreversible polymer of formula I: generating a polymer of TIFF2024521148000080.tif40128; During the ceremony, a, b, and c are mole fractions of comonomers, each of a, b, and c being greater than zero; PEG n is a polyethylene glycol polymer, and n is an integer from 1 to 2500.

[0103] X is independently selected from C, O, and NH. In some examples, X is O. In some examples, X is NH.

[0104] R 1 is alkyl or substituted alkyl; R 2is alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl; and G 1 and G 2 are each independently selected from a polymer segment, an end group, a linker, and an attached modifier. 1 and G 2 is each independently a chain transfer agent. For example, the chain transfer agent may be a dithioester, a dithiocarbamate, a trithiocarbonate, or a xanthate.

[0105] In some cases, n is an integer from 1 to 25, 1 to 50, 1 to 100, 25 to 100, 50 to 100, 1 to 150, 25 to 150, 50 to 150, 100 to 150, 100 to 125, 100 to 150, 150 to 200, 200 to 250, 250 to 500, 500 to 1000, 1000 to 1500, 1500 to 2000, or 2000 to 2500. In some embodiments, the thermoreversible polymer has a variety of molecular weights, such as, for example, 5 kDa to 750 kDa, for example, 10 kDa to 500 kDa, for example, 15 kDa to 450 kDa, for example, 20 kDa to 400 kDa, for example, 25 kDa to 350 kDa, for example, 30 kDa to 300 kDa, for example, 35 kDa to 250 kDa, and for example, 40 kDa to 200 kDa. In certain embodiments, the thermoreversible polymer has a molecular weight of 10 kDa to 500 kDa.

[0106] In some embodiments, R 1 is a C1-C6 alkyl. In some examples, R1 is an alkyl group selected from the group consisting of n-butyl, isobutyl, tert-butyl, n-propyl, pentyl, isopropyl, and isopentyl. In particular examples, R 1 is n-butyl.

[0107] In some embodiments, R 2 is an alkoxy group. In some examples, R 2 is a C1-C6 alkoxy group selected from the group consisting of methoxy, ethoxy, n-propoxy, isoproxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, and isopentoxy. 2 is methoxy.

[0108] In a specific example, a>0.8, 0.2>b>0, and 0.1>c>0.

[0109] In some cases, the method includes copolymerizing N-isopropylacrylamide, N-acryloxysuccinimide, and an alkyl methacrylate to produce a first copolymer comprising an acrylamide backbone, contacting the copolymer with an alkoxypolyethylene glycol amine to produce a second copolymer, and contacting the second copolymer with isopropylamine to produce a copolymer of Formula I: generating a polymer of TIFF2024521148000081.tif40128; During the ceremony, a, b, and c are mole fractions of comonomers, each of a, b, and c being greater than zero; PEG n is a polyethylene glycol polymer, and n is an integer from 1 to 2500.

[0110] R 1 is alkyl or substituted alkyl; R 2 is alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl; and G 1 and G 2are each independently selected from a polymer segment, an end group, a linker, and an attached modifier. 1 and G 2 is each independently a chain transfer agent. For example, the chain transfer agent may be a dithioester, a dithiocarbamate, a trithiocarbonate, or a xanthate.

[0111] In some cases, n is an integer from 1 to 25, 1 to 50, 1 to 100, 25 to 100, 50 to 100, 1 to 150, 25 to 150, 50 to 150, 100 to 150, 100 to 125, 100 to 150, 150 to 200, 200 to 250, 250 to 500, 500 to 1000, 1000 to 1500, 1500 to 2000, or 2000 to 2500. In some embodiments, the thermoreversible polymer has a variety of molecular weights, such as, for example, 5 kDa to 750 kDa, for example, 10 kDa to 500 kDa, for example, 15 kDa to 450 kDa, for example, 20 kDa to 400 kDa, for example, 25 kDa to 350 kDa, for example, 30 kDa to 300 kDa, for example, 35 kDa to 250 kDa, and for example, 40 kDa to 200 kDa. In certain embodiments, the thermoreversible polymer has a molecular weight of 10 kDa to 500 kDa.

[0112] In some cases, the alkyl methacrylate is butyl methacrylate.

[0113] In some embodiments, R 1 is a C1-C6 alkyl. In some examples, R1 is an alkyl group selected from the group consisting of n-butyl, isobutyl, tert-butyl, n-propyl, pentyl, isopropyl, and isopentyl. In particular examples, R 1 is n-butyl.

[0114] In some embodiments, R 2 is an alkoxy group. In some examples, R 2is a C1-C6 alkoxy group selected from the group consisting of methoxy, ethoxy, n-propoxy, isoproxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, and isopentoxy. 2 is methoxy.

[0115] In a specific example, a>0.8, 0.2>b>0, and 0.1>c>0.

[0116] In certain embodiments, a method includes copolymerizing N-isopropylacrylamide and N-acryloxysuccinimide to produce a first copolymer comprising an acrylic backbone, contacting the copolymer with butylamine and methoxypolyethylene glycol amine to produce a second copolymer, and contacting the second copolymer with isopropylamine to produce a copolymer of formula II: generating a polymer of formula TIFF2024521148000082.tif42128; During the ceremony, n is 1 to 25, and G 1 and G 2 are each independently selected from a polymer segment, an end group, a linker, and an attached modifier. 1 and G 2 is each independently a chain transfer agent. For example, the chain transfer agent may be a dithioester, a dithiocarbamate, a trithiocarbonate, or a xanthate.

[0117] In some cases, the reaction of N-isopropylacrylamide with N-acryloxysuccinimide contains about 50 to about 95 mol% N-isopropylacrylamide and the remaining mol% (~100 mol%) N-acryloxysuccinimide, as shown in Figure 1. For example, in some cases, the reaction of N-isopropylacrylamide with N-acryloxysuccinimide contains i) about 50 mol% to about 70 mol% N-isopropylacrylamide, and ii) about 50 mol% to about 30 mol% N-acryloxysuccinimide, as shown in Figure 1. As another example, in some cases, the reaction of N-isopropylacrylamide with N-acryloxysuccinimide contains i) about 70 mol% to about 95 mol% N-isopropylacrylamide, and ii) about 30 mol% to about 5 mol% N-acryloxysuccinimide, as shown in Figure 1. As another example, in some cases, the reaction of N-isopropylacrylamide and N-acryloxysuccinimide includes i) about 80 mol % to about 90 mol % N-isopropylacrylamide, and ii) about 20 mol % to about 10 mol % N-acryloxysuccinimide, as shown in Figure 1. In some cases, the reaction of N-isopropylacrylamide and N-acryloxysuccinimide includes i) 70, 75, 80, 85, 90, or 95 mol % N-isopropylacrylamide, and ii) about 30, 25, 20, 15, 10, or 5 mol % N-acryloxysuccinimide, as shown in Figure 1. The reaction can be carried out at a temperature of 40°C to about 80°C (e.g., about 40°C to about 45°C, about 45°C to about 50°C, about 50°C to about 55°C, about 55°C to about 60°C, about 60°C to about 65°C, about 65°C to about 70°C, or about 70°C to about 80°C). The reaction can be carried out for about 18 hours to about 36 hours (e.g., about 18 hours to about 24 hours, e.g., about 24 hours to about 30 hours, or about 30 hours to about 36 hours). Such a reaction produces an intermediate referred to as PNIPAm-co-PNASI in FIG. 1.

[0118] In some cases, as shown in FIG. 1, the reaction of the PNIPAm-co-PNASI intermediate with butylamine and methoxy PEG amine comprises about 5 mol % to about 30 mol % (e.g., about 5 mol %, about 10 mol %, about 15 mol %, about 20 mol %, about 25 mol %, or about 30 mol %) of butylamine and methoxy PEG amine at about 20 wt % to about 50 wt % (e.g., about 20 wt % to about 25 wt %, about 25 wt % to about 30 wt %, about 30 wt % to about 35 wt %, about 35 wt % to about 40 wt %, about 40 wt % to about 45 wt %, or about 45 wt % to about 50 wt % of PNIPAAm (poly(N-isopropylacrylamide)).

[0119] In some cases, the method includes copolymerizing N-isopropylacrylamide and N-acryloxysuccinimide to produce a first copolymer comprising an acrylamide backbone, contacting the copolymer with an alkylamine (e.g., butylamine) and an alkoxypolyethylene glycol amine to produce a second copolymer, contacting the second copolymer with an aminoalkyl methacrylate (e.g., 2-amino methacrylate) to produce a third copolymer, and contacting the third copolymer with isopropylamine to produce a copolymer of Formula IV: generating a polymer of formula TIFF2024521148000083.tif47128; During the ceremony, a, b, c, and d are mole fractions of comonomers, each of a, b, c, and d being greater than zero; PEG n is a polyethylene glycol polymer, and n is an integer from 1 to 2500; R 1 is alkyl or substituted alkyl; R 2is alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl; G 1 and G 2 are each independently selected from a polymer segment, an end group, a linker, and an attached modifier. 1 and G 2 are each independently a chain transfer agent. For example, the chain transfer agent may be a dithioester, a dithiocarbamate, a trithiocarbonate, or a xanthate. In certain embodiments, G 1 is a carboxyl group. In a particular example, G 1 teeth, TIFF2024521148000084.tif13128, where: TIFF2024521148000085.tif2128 is G 1 and the polymer. In certain embodiments, G 2 teeth, TIFF2024521148000086.tif12128, where: TIFF2024521148000087.tif1128 is G 2 and the polymer.

[0120] In some cases, n is an integer from 1 to 25, 1 to 50, 1 to 100, 25 to 100, 50 to 100, 1 to 150, 25 to 150, 50 to 150, 100 to 150, 100 to 125, 100 to 150, 150 to 200, 200 to 250, 250 to 500, 500 to 1000, 1000 to 1500, 1500 to 2000, or 2000 to 2500. In some embodiments, the thermoreversible polymer has a variety of molecular weights, such as, for example, 5 kDa to 750 kDa, for example, 10 kDa to 500 kDa, for example, 15 kDa to 450 kDa, for example, 20 kDa to 400 kDa, for example, 25 kDa to 350 kDa, for example, 30 kDa to 300 kDa, for example, 35 kDa to 250 kDa, and for example, 40 kDa to 200 kDa. In certain embodiments, the thermoreversible polymer has a molecular weight of 10 kDa to 500 kDa.

[0121] In some embodiments, R 1 is a C1-C6 alkyl. In some examples, R1 is an alkyl group selected from the group consisting of n-butyl, isobutyl, tert-butyl, n-propyl, pentyl, isopropyl, and isopentyl. In particular examples, R 1 is n-butyl.

[0122] In some cases, the method includes copolymerizing N-isopropylacrylamide and N-acryloxysuccinimide with a RAFT agent (e.g., a DMP RAFT agent) to produce a first copolymer comprising an acrylamide backbone, contacting the copolymer with an alkylamine and an alkoxypolyethylene glycol amine to produce a second copolymer, and contacting the second copolymer with isopropylamine to produce a copolymer of Formula I: TIFF2024521148000088.tif40128, During the ceremony, a, b, and c are mole fractions of comonomers, each of a, b, and c being greater than zero; PEG nis a polyethylene glycol polymer, and n is an integer from 1 to 2500; R 1 is alkyl or substituted alkyl; R 2 is alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl; and In certain embodiments, G 1 is a carboxyl group. In a particular example, G 1 teeth, TIFF2024521148000089.tif13128, where: TIFF2024521148000090.tif2128 is G 1 and the polymer. In certain embodiments, G 2 teeth, TIFF2024521148000091.tif12128, where: TIFF2024521148000092.tif2128 is G 2 and the polymer.

[0123] In some cases, n is an integer from 1 to 25, 1 to 50, 1 to 100, 25 to 100, 50 to 100, 1 to 150, 25 to 150, 50 to 150, 100 to 150, 100 to 125, 100 to 150, 150 to 200, 200 to 250, 250 to 500, 500 to 1000, 1000 to 1500, 1500 to 2000, or 2000 to 2500. In some embodiments, the thermoreversible polymer has a variety of molecular weights, such as, for example, 5 kDa to 750 kDa, for example, 10 kDa to 500 kDa, for example, 15 kDa to 450 kDa, for example, 20 kDa to 400 kDa, for example, 25 kDa to 350 kDa, for example, 30 kDa to 300 kDa, for example, 35 kDa to 250 kDa, and for example, 40 kDa to 200 kDa. In certain embodiments, the thermoreversible polymer has a molecular weight of 10 kDa to 500 kDa.

[0124] In some embodiments, R 1 is a C1-C6 alkyl. In some examples, R1 is an alkyl group selected from the group consisting of n-butyl, isobutyl, tert-butyl, n-propyl, pentyl, isopropyl, and isopentyl. In particular examples, R 1 is n-butyl.

[0125] In some embodiments, R 2 is an alkoxy group. In some examples, R 2 is a C1-C6 alkoxy group selected from the group consisting of methoxy, ethoxy, n-propoxy, isoproxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, and isopentoxy. 2 is methoxy.

[0126] In a specific example, a>0.8, 0.2>b>0, and 0.1>c>0.

[0127] composition The present disclosure provides a composition comprising two or more thermoreversible polymers of the present disclosure.In some cases, the composition comprises a mixture of a low molecular weight thermoreversible polymer (e.g., having a molecular weight of 100 kDa or less, such as 75 kDa or less, or 50 kDa or less) and a high molecular weight thermoreversible polymer (e.g., having a molecular weight of 100 kDa or more, such as 200 kDa or more, 300 kDa or more, 500 kDa or more).

[0128] An embodiment of the present disclosure includes a hydrogel composition comprising a) a thermoreversible polymer of the present disclosure, and b) an aqueous solution, such as a buffered aqueous solution. When the hydrogel composition is below its sol-gel transition temperature, the composition can be a homogenous solution and cells present in the solution can be easily removed (e.g., by centrifugation). When the hydrogel composition exceeds its sol-gel transition temperature, the thermoreversible polymer provides a three-dimensional matrix for use in incubation, proliferation, and / or differentiation of cells of interest.

[0129] Any convenient aqueous buffer solution used for incubation and / or differentiation of cells of interest may be utilized in the subject hydrogel compositions. The aqueous buffer solution may contain any convenient component of interest.

[0130] In some examples, the hydrogel composition further comprises a cell of interest (e.g., as described herein). In certain embodiments, the hydrogel composition comprises a stem cell selected from the group consisting of: (a) adult stem cells derived from bone marrow, umbilical cord tissue, or placenta, (b) neural stem cells, and (c) embryonic stem cells. In some cases, the cell is an immune cell, such as a T cell, a natural killer cell, etc. In some cases, the cell is genetically modified with one or more nucleic acids. For example, a T cell can be genetically modified with a nucleic acid comprising a nucleotide sequence encoding a chimeric antigen receptor.

[0131] In certain examples, the thermoreversible polymer is a solid, semi-solid, or gel at or above 20° C., e.g., at or above 21° C., at or above 22° C., at or above 23° C., at or above 24° C., at or above 25° C., at or above 26° C., at or above 27° C., at or above 28° C., at or above 29° C., at or above 30° C., at or above 31° C., at or above 32° C., at or above 33° C., at or above 34° C., at or above 35° C., at or above 36° C., or above. In certain embodiments, the thermoreversible polymer is a solid at 37° C.

[0132] Thermoreversible polymer-cell compositions The present disclosure provides a composition comprising a) a thermoreversible polymer of the present disclosure, and b) cells embedded or suspended within the polymer. The thermoreversible polymer-cell composition of the present disclosure is useful for generating a desired number of cells by culturing the thermoreversible polymer-cell composition under conditions and for a period of time sufficient to generate a desired number of cells. Such cells may include stem cells, differentiated cells, and the like. The thermoreversible polymer-cell composition of the present disclosure is useful for differentiating cells to, for example, generate a desired number of differentiated cells. The thermoreversible polymer-cell composition of the present disclosure can be implanted into an individual in need thereof, where the cells proliferate and / or differentiate within the implanted thermoreversible polymer-cell composition and migrate out of the implanted thermoreversible polymer-cell composition.

[0133] Methods for culturing cells The thermoreversible polymers of the present disclosure can be used to culture cells in vitro or in vivo. Thus, the present disclosure provides a method of culturing cells, which includes contacting cells with a thermoreversible polymer and culturing the thermoreversible polymer containing the cells under conditions suitable for cell growth and / or differentiation. In some cases, the methods of the present disclosure include culturing cells contained (e.g., embedded, suspended, etc.) within the hydrogel compositions of the present disclosure.

[0134] In some cases, the disclosed methods for culturing cells include culturing cells in a hydrogel composition of the present disclosure at a temperature where the hydrogel composition is semi-solid (e.g., a gel) (e.g., about 30° C. to about 37° C., e.g., 37° C.). In some cases, the disclosed methods for culturing cells include culturing cells in a hydrogel composition of the present disclosure at a temperature where the hydrogel composition is liquid (e.g., about 4° C. to about 10° C., e.g., 4° C.).

[0135] The disclosed methods for culturing cells can be used to generate a desired number of cells, including differentiated cells and stem cells. For example, the disclosed methods can be used to culture 10 2 cells ~ about 10 9 Cells, e.g., about 10 2 Cells ~ approx. 5 x 10 2 cells, approximately 5 x 10 2 cells ~ about 10 3 cells, about 10 3 Cells ~ approx. 5 x 10 3 cells, approximately 5 x 10 3 cells ~ about 10 4 cells, about 10 4 Cells ~ approx. 5 x 10 4 cells, approximately 5 x 10 4 cells ~ about 10 5 cells, about 10 5 Cells ~ approx. 5 x 10 5 cells, approximately 5 x 10 5 cells ~ about 10 6 cells, about 10 6 Cells ~ approx. 5 x 10 6 cells, approximately 5 x 10 6 cells ~ about 10 7 cells, about 10 7 Cells ~ approx. 5 x 10 7 cells, approximately 5 x 10 7 cells ~ about 10 8 cells, about 10 8 Cells ~ approx. 5 x 10 8 cells, or approximately 5 x 10 8 cells ~ about 10 9 In some cases, cells can be produced using the methods of the present disclosure. 9 More than 10 cells, e.g. 9 cells ~5×109 cells, 5 x 10 9 cells ~10 10 cells, 10 10 cells ~5×10 10 cells, 5 x 10 10 cells ~10 11 cells, 10 11 cells ~5×10 11 cells, 5 x 10 11 cells ~10 12 cells, 10 12 cells ~5×10 12 cells, 5 x 10 12 cells ~10 13 cells, 10 13 cells ~5×10 13 cells, 5 x 10 13 cells ~10 14 cells, 10 14 cells ~5×10 14 cells, or 5 × 10 14 cells ~10 15 The cells can be generated.

[0136] Cells can be cultured in the hydrogel compositions of the present disclosure, ranging from 10 cells / mL (or cubic centimeter) of hydrogel to about 10 8 cells / mL, for example, about 10 cells / mL to about 10 2 cells / mL, approximately 10 2 cells / mL~about 10 4 cells / mL, approximately 10 4 cells / mL~about 10 6 cells / mL, or approximately 10 6 cells / mL~about 10 8 The cells can be present in the hydrogel composition (eg, embedded in the hydrogel composition, suspended in the hydrogel composition, etc.) at a density of cells / mL.

[0137] In some cases, the hydrogel composition maintains the pluripotency of the pluripotent stem cells contained within the hydrogel composition. For example, in some cases, the hydrogel composition maintains the pluripotency of the pluripotent stem cells contained within the hydrogel composition when cultured in the hydrogel composition for a period of 1 day to 6 months or more. For example, in some cases, the hydrogel composition maintains the pluripotency of the pluripotent stem cells contained within the hydrogel composition when cultured in the hydrogel composition for a period of 1 day to 7 days, 1 week to 2 weeks, 2 weeks to 1 month, 1 month to 2 months, 2 months to 4 months, or 4 months to 6 months. For example, in some cases, the hydrogel composition maintains the pluripotency of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 90% of the pluripotent stem cells contained in the hydrogel composition when cultured in the hydrogel composition for a period of 1 day to 7 days, 1 week to 2 weeks, 2 weeks to 1 month, 1 month to 2 months, 2 months to 4 months, or 4 months to 6 months. In some cases, the hydrogel composition provides sufficient time for cell proliferation. In some cases, the cells cultured in the hydrogel composition maintain pluripotency after 1 passage, 2 passages, 3 passages, or more than 3 passages. In some cases, the hydrogel composition maintains the pluripotency of human pluripotent stem cells (hPSCs). In some cases, the hPSCs are H1 embryonic stem cells (H1ESCs). In some cases, the hPSCs are H9 embryonic stem cells (H9ESCs). In some cases, the hydrogel composition maintains the pluripotency of induced pluripotent stem cells (iPSCs). In some cases, iPSCs cultured in the hydrogel maintain pluripotency after 1 passage, 2 passages, 3 passages, or more than 3 passages (e.g., after 4 passages, after 5 passages, after 5-10 passages, after 10-15 passages, after 15-20 passages, etc.).

[0138] In some cases, the cells cultured in the hydrogel composition aggregate. For example, in some cases, the cells cultured in the hydrogel composition grow as small aggregates after 1 day of culture. In some cases, the cells cultured in the hydrogel composition grow as single cells after 1 day of culture. In some cases, the cells cultured in the hydrogel composition aggregate after 2 days of culture. In some cases, the cells cultured in the hydrogel composition aggregate after 3 days of culture. In some cases, the cells cultured in the hydrogel composition aggregate after 4 days of culture. In some cases, the cells are H9ESC. In some cases, the H9ESC grow as small aggregates after 1 day of culture. In some cases, the H9ESC grow as large aggregates after 4 days of culture.

[0139] The hydrogel composition may include one or more factors (e.g., polypeptides, small molecules, etc.) that promote the proliferation or differentiation of cells cultured in the hydrogel composition. Suitable factors include, for example, retinoic acid, Wnt agonists, Shh signaling pathway agonists, bone morphogenetic protein (BMP) inhibitors (e.g., Noggin), receptor tyrosine kinase ligands (e.g., epidermal growth factor), nicotinamide, p38 inhibitors, dual Smad inhibitors, Rock inhibitors, gastrin, activators of prostaglandin signaling pathways, fibroblast growth factors (FGF) (e.g., FGF10), TGF-β inhibitors, Rspondin, Rspondin mimetics, and combinations of two or more of the aforementioned factors. Such factors may be present in the hydrogel composition at a concentration ranging from 1 nM to 100 mM, e.g., 1 nM to 50 nM, 50 nM to 100 nM, 100 nM to 0.5 μM, 0.5 μM to 1 μM, 1 μM to 50 μM, 50 μM to 100 μM, 100 μM to 0.5 mM, 0.5 μM to 1 mM, 1 mM to 50 mM, or 50 mM to 100 mM. Such factors may be present in the hydrogel composition at a concentration ranging from 1 ng / ml to 1 mg / ml, e.g., 1 ng / ml to 50 ng / ml, 50 ng / ml to 100 ng / ml, 100 ng to 0.5 μg / ml, 0.5 μg / ml to 1 μg / ml, 1 μg / ml to 50 μg / ml, 50 μg / ml to 100 μg / ml, 100 μg / ml to 500 μg / ml, 500 μg / ml to 0.1 mg / ml, 0.1 mg / ml to 0.5 mg / ml, or 0.5 mg / ml to 1 mg / ml, or greater than 1 mg / ml.

[0140] In some cases, the hydrogel compositions of the present disclosure comprise one or more of Rspondin 1-4 and / or Rspondin mimetics, BMP inhibitors (e.g., noggin), TGF-β inhibitors, receptor tyrosine kinase ligands (e.g., EGF), nicotinamide, Wnt agonists (e.g., Wnt(3a)), Wnt antagonists (e.g., IWP-2, IWP-3, IWP-4, Dkk1, etc.), p38 inhibitors, gastrin, FGF10, HGF, and ROCK inhibitors.

[0141] IWP2 has the following structure: I have TIFF2024521148000093.tif45128.

[0142] Several classes of natural BMP binding proteins are known, including noggin, chordin, and chordin-like proteins including chordin domains, follistatin, and follistatin-related proteins including follistatin domains, DAN, and DAN-like proteins including DAN cysteine ​​knot domains, sclerostin / SOST, and α2 macroglobulin. BMP inhibitors are agents that bind to BMP molecules to form complexes, for example, reducing BMP activity by preventing or inhibiting the binding of BMP molecules to BMP receptors. Alternatively, inhibitors may be agents that bind to BMP receptors and prevent the binding of BMP ligands to the receptor, for example, antibodies that bind to the receptor. BMP inhibitors may be proteins or small molecules, and may be naturally occurring, modified, and / or partially or fully synthetic. BMP inhibitors may be noggin, DAN, or DAN-like proteins including Cerberus and Gremlin. In some cases, the BMP inhibitor is noggin. BMP inhibitors (e.g., noggin) may be used at any suitable concentration. The hydrogel compositions of the present disclosure may contain Noggin at a concentration of about 10 ng / ml to about 100 ng / ml.

[0143] The hydrogel compositions of the present disclosure may include one or more Wnt agonists. The Wnt signaling pathway is defined by a series of events that occur when a Wnt protein binds to a cell surface receptor of a Frizzled receptor family member. This activates Dishevelled family proteins that inhibit a complex of proteins including axin, GSK-3, and the protein APC to degrade intracellular β-catenin. The resulting enriched nuclear β-catenin enhances transcription by TCF / LEF family transcription factors. A Wnt agonist is defined as an agent that activates TCF / LEF-mediated transcription in cells. Wnt agonists can be Frizzled receptor family members, including any and all Wnt family proteins, inhibitors of intracellular β-catenin degradation, and Wnt agonists that bind to and activate activators of TCF / LEF.

[0144] Suitable Wnt agonists include secreted glycoproteins, including Wnt-1 / Int-1, Wnt-2 / Irp (InM-related protein), Wnt-2b / 13, Wnt-3 / Int-4, Wnt-3a, Wnt-4, Wnt-5a, Wnt-5b, Wnt-6, Wnt-7a, Wnt-7b, Wnt-8a / 8d, Wnt-8b, Wnt-9a / 14, Wnt-9b / 14b / 15, Wnt-10a, Wnt-10b / 12, WnM1, and Wnt-16. Other suitable Wnt agonists include the R-spondin family of secreted proteins, which are involved in the activation and regulation of the Wnt signaling pathway and are composed of four members (R-spondin1, R-spondin2, R-spondin3, and R-spondin4), and Norrin (also called Norm disease protein or NDP) is a secreted regulatory protein that functions like Wnt protein in that it binds with high affinity to Frizzled-4 receptor and induces the activation of the Wnt signaling pathway.Also suitable are R-spondin mimics, such as agonists of Lgr5, such as anti-Lgr5 antibodies.

[0145] Suitable Wnt agonists include glycogen synthase kinase-3 (GSK-3) inhibitors.Known GSK-3 inhibitors include small interfering RNA (siRNA), lithium, kenpaullone, 6-bromoindirubin-30-acetoxime, SB216763 and SB415286, and FRAT family members and FRAT-derived peptides that interfere with the interaction of GSK-3 with axin.

[0146] Suitable Wnt agonists include Wnt-3a, GSK-3 inhibitors (such as CHIR99021), Wnt5, Wnt-6a, Norrin, and any other Wnt family protein.

[0147] The Wnt agonist may be included in the hydrogel composition at a suitable concentration, for example, CHIR99021 (6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile) may be included at a final concentration of 50 nM to 100 μM, for example 100 nM to 50 μM, 1 μM to 10 μM, 1 μM to 5 μM, or 3 μM.

[0148] Exemplary GSK-3 inhibitors include CHIR99021 (6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile, CAS number: 252917-06-9), SB-216763 (3-(2,4-dichlorophenyl)-4-(1-methyl-1H-ind-3-yl)-1H-pyrrole-2,5-dione, CAS number: 280744-09-4), 6-bromoindirubin-3'-oxime (CAS number: CAS667463-62-9), tideglusib (4-benzyl-2-(naphthalen-1-yl)-1,2,4-thiadiazolidine-3,5-dione), GSK- 3 inhibitor 1 (CAS number: 603272-51-1), AZD1080 (CAS number: 612487-72-6), TDZD-8 (4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione, CAS number: 327036-89-5), TWS119 (3-[[6-(3-aminophenyl)-7H-pyrrolo[2,3-d]pyrimidine- 4-yl]oxy]-phenol, CAS number: 601514-19-6), CHIR-99021 (CAS number: 252917-06-9), CHIR-98014 (N6-[2-[[4-(2,4-dichlorophenyl)-5-(1H-imidazol-1-yl)-2-pyrimidinyl]amino]-ethyl]-3-nitro-2,6-pyridinediamine;CAS number: 252935-94-7), SB415286 (3-[(3-chloro-4-hydroxyphenyl)-amino]-4-(2-nitrophenyl)-1H-pyrrole-2,5-dione, CAS number: 264218-23-7), LY2090314 (3-(9-fluoro-2-(piperidine-1-carbonyl)-1,2,3,4-tetrahydro-[1,4]diazepino[-6,7,1-hi]indol-7-yl)-4-(imidazo[1,2-a]pyridin-3-yl)-1H-pyrrole-2,5-dione, , CAS number: 603288-22-8), AR-A014418 (N-(4-methoxybenzyl)-N'-(5-nitro-1,3-thiazol-2-yl)urea, CAS number: 487021-52-3 and / or IM-12 (3-(4-fluorophenylethylamino)-1-methyl-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione, CAS number: 1129669-05-1). Thus, the GSK-3 inhibitor can also be CHIR99021.;

[0149] The hydrogel composition of the present disclosure may include one or more receptor tyrosine kinase ligands.An example of a suitable receptor tyrosine kinase ligand is EGF, which is the ligand for the receptor tyrosine kinase EGFR.Many receptor tyrosine kinase ligands are also mitogenic growth factors.

[0150] The hydrogel composition of the present disclosure may include a TGF-β inhibitor. Examples of suitable TGF-β inhibitors include, for example, 3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide (A83-01), 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide (SB-431542), and the like. Suitable TGF-β inhibitors include those listed in Table 1 of US Patent Publication No. 2014 / 0243227, including, for example, A83-01, SB-431542, SB-505124, SB-525334, SD-208, LY-36494, and SJN-2511.

[0151] The hydrogel compositions of the present disclosure may include one or more mitotic growth factors selected from the growth factor family including epidermal growth factor (EGF), transforming growth factor alpha (TGF-α), basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF), and keratinocyte growth factor (KGF).

[0152] The hydrogel composition of the present disclosure may include a Rock (Rho kinase) inhibitor. Suitable Rock inhibitors include, for example, R-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632, Sigma-Aldrich), 5-(1,4-diazepan-1-ylsulfonyl)isoquinoline (Fasudil or HA1077, Cayman Chemical), and (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine dihydrochloride (H-1 152, Tocris Bioschience).

[0153] The hydrogel composition of the present disclosure may include a Notch agonist. Examples of suitable Notch agonists include Jagged 1 and Delta 1, or active fragments or derivatives thereof. A suitable Notch agonist is the DSL peptide (Dontu et al., 2004. Breast Cancer Res 6.R605-R615) having the sequence CDDYYYGFGCNKFCRPR (SEQ ID NO:1).

[0154] The hydrogel compositions of the present disclosure may include activators of the prostaglandin signaling pathway, such as, for example, phospholipids, arachidonic acid (AA), prostaglandin E2 (PGE2), prostaglandin G2 (PGG2), prostaglandin F2 (PGF2), prostaglandin H2 (PGH2), and prostaglandin D2 (PGD2).

[0155] The hydrogel compositions of the present disclosure may include a RANK ligand.

[0156] The pH of the hydrogel composition of the present disclosure may be in the range of about 7.0 to 7.8, about 7.2 to 7.6, or about 7.4. The pH may be maintained using a buffer. A suitable buffer can be readily selected by one of ordinary skill in the art. Buffers that may be used include carbonate buffers (e.g., NaHCO3) and phosphate buffers (e.g., NaH2PO4). Other buffers such as N-[2-hydroxyethyl]-piperazine-N-[2-ethanesulfonic acid] (HEPES) and 3-[N-morpholino]-propanesulfonic acid (MOPS) may also be used.

[0157] The hydrogel compositions of the present disclosure may include one or more amino acids, which may be present include L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-cystine, L-glutamic acid, L-glutamine, L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, and combinations thereof.

[0158] The hydrogel compositions of the present disclosure may include one or more vitamins. Vitamins that may be present include thiamine (vitamin B1), riboflavin (vitamin B2), niacin (vitamin B3), D-calcium pantothenate (vitamin B5), pyridoxal / pyridoxamine / pyridoxine (vitamin B6), folic acid (vitamin B9), cyanocobalamin (vitamin B12), ascorbic acid (vitamin C), calciferol (vitamin D2), DL-alpha tocopherol (vitamin E), biotin (vitamin H), and menadione (vitamin K).

[0159] The hydrogel composition of the present disclosure may include one or more inorganic salts. Inorganic salts that may be present include calcium, copper, iron, magnesium, potassium, sodium, and zinc salts. Salts are usually used in the form of chlorides, phosphates, sulfates, nitrates, and bicarbonates.

[0160] In some cases, the hydrogel compositions of the present disclosure do not include serum, e.g., the hydrogel compositions are serum-free. In some cases, the hydrogel compositions of the present disclosure include a serum substitute.

[0161] The hydrogel compositions of the present disclosure may include other components. The hydrogel compositions of the present disclosure may include standard media components such as amino acids, vitamins, inorganic salts, carbon energy sources, and buffers. Other standard cell culture components that may be included in the culture include hormones such as progesterone, proteins such as albumin, catalase, insulin, and transferrin.

[0162] The hydrogel composition of the present disclosure may include known cell culture media. Those skilled in the art will understand from general knowledge the types of media that can be used for cell culture, including stem cell culture. Suitable cell culture media are commercially available and include, but are not limited to, Dulbecco's Modified Eagle Medium (DMEM), Minimal Essential Medium (MEM), Knockout DMEM (KO-DMEM), Glasgow Minimal Essential Medium (G-MEM), Basal Eagle Medium (BME), DMEM / Ham's F12 medium, Advanced DMEM / Ham's F12 medium, Iscove's Modified Dulbecco's Medium, and Minimal Basal Medium (MEM), Ham's F-10 medium, Ham's F-12 medium, 199 medium, and RPMI 1640 medium.

[0163] Cells that can be cultured using the methods of the present disclosure include mammalian cells. The cells can be undifferentiated cells, such as pluripotent, multipotent, oligopotent, or unipotent cells. The cells can be differentiated cells. The cells can be a mixture of differentiated and undifferentiated cells. The cells cultured in the hydrogel compositions of the present disclosure can be a single type of cell or a mixture of two or more types of cells.

[0164] The cells may be primary cells, genetically modified cells (e.g., genetically modified primary cells), and the like. The cells may be human cells, non-human primate cells, rodent (e.g., mouse, rat) cells, lagomorph (e.g., rabbit) cells, ungulate cells, and the like. Cells of any of a variety of cell types can be cultured using the methods of the present disclosure. Such cells may include cells from tissue samples including, but not limited to, blood, bone, brain, kidney, muscle, spinal cord, nerve, endocrine system, uterus, ear, foreskin, liver, intestine, bladder, or skin. The cells may be obtained from an individual suffering from a particular disease or an individual in need of pluripotent stem cells. The cells may include nerve cells, lymphocytes, epidermal cells, intestinal cells, fibroblasts, keratinocytes, adipocytes, cardiomyocytes, pancreatic islet cells, hepatocytes, astrocytes, oligodendrocytes, retinal cells, and the like. The cells may be autologous, e.g., the cells may be obtained from an individual, cultured using the methods of the present disclosure, and then, after culture (and possible modification, differentiation, etc.), returned to the individual from whom the cells were obtained. In some cases, the cells are human cells. In some cases, the cells are rodent (e.g., mouse, rat) cells. In some cases, the cells are non-human primate cells.

[0165] stem cells Cells that can be cultured using the methods of the present disclosure include hematopoietic stem cells, embryonic stem cells, mesenchymal stem cells, neural stem cells, epidermal stem cells, endothelial stem cells, gastrointestinal stem cells, hepatic stem cells, umbilical cord blood stem cells, amniotic fluid stem cells, skeletal muscle stem cells, smooth muscle stem cells (e.g., cardiac smooth muscle stem cells), pancreatic stem cells, olfactory stem cells, hematopoietic stem cells, induced pluripotent stem cells, and the like.

[0166] In some cases, the cells cultured using the methods of the present disclosure are stem cells. In some cases, the cells cultured using the methods of the present disclosure are pluripotent stem cells.

[0167] Suitable human embryonic stem (ES) cells include various available human ES lines, e.g., BG01 (hESBGN-01), BG02 (hESBGN-02), BG03 (hESBGN-03) (BresaGen, Inc.; Athens, Ga.); SA01 (Sahlgrenska 1), SA02 (Sahlgrenska 2) (Cellartis AB; Goeteborg, Sweden); ES01 (HES-1), ES01 (HES-2), ES03 (HES-3), ES04 (HES-4), ES05 (HES-5), ES06 (HES-6) (ES Cell International; Singapore); UC01 (HSF-1), UC06 (HSF-6) (University of California, San Francisco; San Francisco, CA). Francisco, Calif.); WA01(H1), WA07(H7), WA09(H9), WA13(H13), WA14(H14) (Wisconsin Alumni Research Foundation; WARF; Madison, Wis.). Cell line designations are based on the National Institutes of Health (NIH) code followed by the donor code in parentheses. See, e.g., U.S. Patent No. 6,875,607. Suitable human ES cell lines may be positive for one, two, three, four, five, six, or all seven of the following markers: stage-specific embryonic antigen 3 (SSEA-3), SSEA-4, TRA 1-60, TRA 1-81, Oct-4, GCTM-2, and alkaline phosphatase.

[0168] Hematopoietic stem cells (HSCs) are mesoderm-derived cells that can be isolated from bone marrow, blood, umbilical cord blood, fetal liver, and the yolk sac. HSCs express the CD34 + and CD3 -HSCs are characterized as being capable of repopulating erythroid, neutrophil-macrophage, megakaryocyte, and lymphoid hematopoietic cell lineages in vivo. In vitro, HSCs can be induced to undergo at least some self-renewal cell division and can be induced to differentiate into the same lineages as those found in vivo. Thus, HSCs can be induced to differentiate into one or more of erythroid cells, megakaryocytes, neutrophils, macrophages, and lymphocytes.

[0169] Neural stem cells (NSCs) can differentiate into neurons and glia (including oligodendrocytes and astrocytes). Neural stem cells are pluripotent stem cells capable of multiple divisions and can generate, under certain conditions, daughter cells that are neural stem cells or neural progenitor cells that can be neuroblasts or glioblasts, for example, cells committed to becoming one or more types of neurons and glial cells, respectively. Methods for obtaining NSCs are known in the art. In some cases, NSCs cultured in hydrogel compositions maintain pluripotency even after multiple passages.

[0170] Mesenchymal stem cells (MSCs) are originally derived from fetal mesoderm and can be isolated from adult bone marrow and differentiate to form muscle, bone, cartilage, fat, bone marrow stroma, and tendon.Methods for isolating MSCs are known in the art, and any known method can be used to obtain MSCs.See, for example, U.S. Patent No. 5,736,396, which describes the isolation of human MSCs.

[0171] Induced pluripotent stem (iPS) cells are pluripotent stem cells derived from somatic cells, e.g., differentiated somatic cells. iPS cells can self-renew and differentiate into committed stem cells, including neural stem cells, and various types of mature cells.

[0172] iPS cells can be generated from somatic cells, including skin fibroblasts, for example, using known methods. iPS cells produce and express on their cell surface one or more of SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49 / 6E, and Nanog cell surface antigens. In some embodiments, iPS cells produce and express on their cell surface SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49 / 6E, and Nanog. iPS cells express one or more of Oct-3 / 4, Sox2, Nanog, GDF3, REX1, FGF4, ESG1, DPPA2, DPPA4, and hTERT genes. In some embodiments, the iPS cells express Oct-3 / 4, Sox2, Nanog, GDF3, REX1, FGF4, ESG1, DPPA2, DPPA4, and hTERT. Methods for generating iPS are known in the art, and any such method can be used to generate iPS. See, e.g., Takahashi and Yamanaka (2006) Cell 126:663-676; Yamanaka et.al. (2007) Nature 448:313-7; Wernig et al. (2007) Nature 448:318-24; Maherali (2007) Cell Stem Cell 1:55-70; Nakagawa et al. (2008) Nat. Biotechnol. 26:101; Takahashi et al. (2007) Cell 131:861; Takahashi et al. (2007) Nat. Protoc. 2:3081; and Okita et al. (2007 Nature 448:313).

[0173] iPS cells can be generated from somatic cells (e.g., skin fibroblasts) by genetically modifying the somatic cells with one or more expression constructs encoding Oct-3 / 4 and Sox2. In some embodiments, the somatic cells are genetically modified with one or more expression constructs comprising nucleotide sequences encoding Oct-3 / 4, Sox2, c-myc, and Klf4. In some embodiments, the somatic cells are genetically modified with one or more expression constructs comprising nucleotide sequences encoding Oct-4, Sox2, Nanog, and LIN28.

[0174] In some cases, the cells cultured using the methods of the present disclosure are somatic stem cells (also known as "adult stem cells"). Suitable somatic stem cells include, for example, tissue stem cells and tissue progenitor cells. Stem cells that can be cultured in the hydrogel compositions of the present disclosure include, for example, neural stem cells, hematopoietic stem cells, mammary stem cells, epidermal stem cells, intestinal stem cells, mesenchymal stem cells, endothelial stem cells, pancreatic stem cells, dermal stem cells, cardiac stem cells, oligodendrocyte progenitor cells, neural stem cells, adult olfactory stem cells, neural crest stem cells, hepatic stem cells, and the like.

[0175] immune cells Cells that can be cultured using the methods of the present disclosure include immune cells. Immune cells include, for example, T cells, natural killer (NK) cells, B cells, etc. T cells include CD4 + T cells, CD8 + The cell may be a T cell, a regulatory T cell (Treg), etc. In some cases, the cell is genetically modified with one or more nucleic acids that include a nucleotide sequence that encodes a protein of interest. For example, the T cell may be genetically modified with a nucleic acid that includes a nucleotide sequence that encodes a chimeric antigen receptor.

[0176] Cell Differentiation Methods The present disclosure provides a method for producing differentiated cells from stem or progenitor cells, the method comprising culturing stem or progenitor cells in a hydrogel composition of the present disclosure for a period of time and under conditions suitable for inducing differentiation of the stem or progenitor cells. The conditions for inducing differentiation of stem or progenitor cells depend, in part, on the differentiated cells desired. The conditions include including one or more factors in the hydrogel that induce differentiation.

[0177] Cell Isolation Methods The present disclosure provides a method for producing stem cells, progenitor cells, or differentiated cells, the method comprising a) culturing cells in a hydrogel composition of the present disclosure, and b) isolating the cells from the hydrogel composition. For example, in some cases, cells are cultured in a hydrogel composition of the present disclosure at a temperature where the hydrogel is semi-solid (e.g., gel) (e.g., 37°C), and the cells or the progeny of the cells are isolated from the hydrogel composition by lowering the temperature of the hydrogel composition (e.g., to about 4°C) so that the hydrogel composition becomes liquid. The cells can be isolated from the hydrogel composition in liquid form using centrifugation or any other means.

[0178] In some cases, the methods of the present disclosure include a) culturing stem cells in a hydrogel composition of the present disclosure at a temperature where the hydrogel is semi-solid (e.g., a gel), where the hydrogel composition comprises one or more factors that induce differentiation of the stem cells, b) reducing the temperature of the hydrogel composition such that it becomes a liquid, and c) isolating the differentiated cells from the liquid.

[0179] In some cases, the methods of the present disclosure include a) culturing stem cells in a hydrogel composition of the present disclosure at a temperature where the hydrogel is semi-solid (e.g., a gel), where the hydrogel composition comprises one or more factors that promote the growth and proliferation of stem cells, b) reducing the temperature of the hydrogel composition such that the hydrogel composition becomes a liquid, and c) isolating the expanded stem cells from the liquid.

[0180] Treatment method The present disclosure provides methods of treating a disease or disorder in an individual in need of such treatment. In some cases, the methods include culturing cells using the methods of the present disclosure, isolating the cells, and administering the isolated cells to the individual, as described above. In some cases, the methods include implanting a thermoreversible polymer-cell composition of the present disclosure into the individual.

[0181] Diseases that can be treated using cells cultured in the thermoreversible polymer of the present disclosure or using the thermoreversible polymer-cell composition of the present disclosure include, but are not limited to, autoimmune diseases, diseases in which the treatment involves regeneration of nerve cells / tissues, diseases in which the treatment involves regeneration of cardiac cells / tissues, Parkinson's disease, and Alzheimer's disease. Cells differentiated from stem cells using the methods of the present disclosure include cardiomyocytes, insulin-producing cells, nerve cells, oligodendrocytes, etc., and such cells can be safely utilized in stem cell transplantation therapy for the treatment of various diseases such as heart failure, insulin-dependent diabetes, Parkinson's disease, and spinal cord injury. Stem cells, or differentiated cells derived therefrom, can be used for autologous cell therapy, which therapy is specific (e.g., individualized) to a particular subject. Stem cells, or differentiated cells derived therefrom, can be used for non-autologous therapy.

[0182] Suitable subjects for treatment with the subject methods include individuals diagnosed with a blood cell cancer (e.g., leukemia), individuals diagnosed with AIDS, individuals with sickle cell anemia, individuals with immune disorders such as acquired immune deficiency, inherited immune deficiency, individuals with type 1 diabetes, individuals with nervous system disorders such as Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, spinal cord injury, stroke, hepatitis, cirrhosis, metabolic disorders affecting the liver or central nervous system (e.g., lysosomal storage diseases), etc. These include individuals with liver disorders, individuals with cartilage or bone disorders, such as individuals requiring joint replacement surgery, individuals with osteoarthritis, individuals with osteoporosis, individuals suffering from cardiac disorders, such as myocardial infarction, coronary artery disease, or other disorders resulting in ischemic cardiac tissue, individuals suffering from renal disorders, such as renal failure (e.g., individuals undergoing renal dialysis), individuals with skeletal muscle disorders, such as muscular dystrophies, and individuals with pulmonary disorders, such as emphysema, pulmonary fibrosis, idiopathic pulmonary fibrosis.

[0183] usefulness The subject thermoreversible polymers, hydrogels and methods find use in a variety of applications. Applications of interest include, but are not limited to, applications in which cell culture and / or differentiation are of interest. Protocols of interest can use single cells or small aggregates of stem cells, which can be uniformly dispersed throughout the hydrogel material at low temperatures. The material can then be spread onto a two-dimensional surface or dropped into warm medium in a stirred tank reactor. Upon warming to 37°C, the material gels and encapsulates the cells. After changing the medium daily or every other day and checking the progress of cell growth, the material can be cooled and centrifuged to isolate the cells.

[0184] Examples of Non-Limiting Aspects of the Disclosure The aspects (including embodiments) of the subject matter described above may be beneficial alone or in combination with one or more other aspects or embodiments. Without limiting the foregoing, certain non-limiting aspects of the present disclosure are provided below. As will be apparent to one of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the individually numbered aspects described above or below. This is intended to provide support for all such combinations of aspects, and is not limited to the combinations of aspects expressly provided below.

[0185] Aspect 1. A thermoreversible polymer comprising a) N-isopropylacrylamide (NIPAM) comonomer, and b) a lower alkylamine comonomer, and c) a poly(ethylene glycol) (PEG) comonomer, The thermoreversible polymer, wherein the terminal PEG monomer is substituted with alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl.

[0186] Embodiment 2. The thermoreversible polymer of embodiment 1, wherein i) the lower alkylamine comonomer comprises n-butyl, isobutyl, tert-butyl, n-propyl, pentyl, isopropyl, or isopentyl, and ii) the terminal PEG monomer is substituted with an alkoxy group.

[0187] Aspect 3. The thermoreversible polymer of aspect 2, wherein the alkoxy group is a C1 to C6 alkoxy selected from the group consisting of methoxy, ethoxy, n-propoxy, isoproxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, and isopentoxy.

[0188] Aspect 4. Formula (I): TIFF2024521148000094.tif40128 included, During the ceremony, a, b, and c are mole fractions of the comonomer, and a, b, and c are each greater than zero; PEG n is a polyethylene glycol polymer, and n is an integer from 1 to 2500; X is independently selected from C, O, or NH; R 1 is alkyl or substituted alkyl; R 2 is alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl; and G 1 and G 2 are each independently selected from a polymer segment, an end group, a linker, and an attached modifying agent; The thermoreversible polymer of any one of embodiments 1 to 3.

[0189] Aspect 5. R 1 is C1-C6 alkyl.

[0190] Aspect 6. R 1 is an alkyl group selected from the group consisting of n-butyl, isobutyl, tert-butyl, n-propyl, pentyl, isopropyl, and isopentyl.

[0191] Aspect 7. R 1 The thermoreversible polymer of embodiment 6, wherein is n-butyl.

[0192] Aspect 8. R 2 The thermoreversible polymer of any one of embodiments 4 to 7, wherein is an alkoxy group.

[0193] Aspect 9. R 2is a C1 to C6 alkoxy group selected from the group consisting of methoxy, ethoxy, n-propoxy, isoproxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, and isopentoxy.

[0194] Aspect 10. R 2 The thermoreversible polymer of embodiment 9, wherein is methoxy.

[0195] Embodiment 11. The thermoreversible polymer of any one of embodiments 2 to 10, wherein a>0.8, 0.2>b>0, and 0.1>c>0.

[0196] Aspect 12. Formula II: TIFF2024521148000095.tif42128 included, During the ceremony, n is 1 to 25, and G 1 and G 2 are each independently selected from a polymer segment, an end group, a linker, and an attached modifying agent; The thermoreversible polymer of any one of embodiments 1 to 11.

[0197] 13. The PEG or PEG n 13. The thermoreversible polymer of any one of embodiments 1-12, wherein said polymer has a molecular weight of from 2 kDa to 100 kDa.

[0198] Aspect 14. Formula (III): TIFF2024521148000096.tif40128 included, During the ceremony, a, b, c, and d are mole fractions of said comonomers, and a, b, c, and d are each greater than zero; PEG n is a polyethylene glycol polymer, and n is an integer from 1 to 2500; R 1 is alkyl or substituted alkyl; R 2is alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl; L is a linker, Z 2 is a modifier, and G 1 and G 2 are each independently selected from a polymer segment, an end group, a linker, and an attached modifying agent; The thermoreversible polymer of any one of embodiments 1 to 3.

[0199] Aspect 15. R 1 is C1-C6 alkyl.

[0200] Aspect 16. R 1 is an alkyl group selected from the group consisting of n-butyl, isobutyl, tert-butyl, n-propyl, pentyl, isopropyl, and isopentyl.

[0201] Aspect 17. R 1 17. The thermoreversible polymer of embodiment 16, wherein is n-butyl.

[0202] Aspect 18. R 2 18. The thermoreversible polymer of any one of embodiments 14-17, wherein is an alkoxy group.

[0203] Aspect 19. R 2 is a C1 to C6 alkoxy group selected from the group consisting of methoxy, ethoxy, n-propoxy, isoproxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, and isopentoxy.

[0204] Aspect 20. R 2 The thermoreversible polymer of embodiment 19, wherein is methoxy.

[0205] Aspect 21. Z 2 is a chemoselective functional group selected from thiols, alkynes, cyclooctynes, azides, phosphines, maleimides, alkoxyamines, aldehydes, and protected forms or precursors thereof.

[0206] Aspect 22. Z 2 is a modifier selected from heparin, hyaluronic acid, a specific binding member, a peptide, a nucleic acid, gelatin, fibronectin, collagen, laminin, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin, progesterone, glucose, stromal cell-derived factor-1 (SDF-1), thymosin beta4, sonic hedgehog (SHH), noggin, activin, transforming growth factor beta (TGF-β) (TGFβ3), FGF8, brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), neurotrophic factor 3 (NT3), platelet-derived growth factor (PDGF), and insulin-like growth factor-1 (IGF-1).

[0207] Embodiment 23. The thermoreversible polymer of any one of embodiments 2 to 10, wherein a>0.8, 0.2>b>0, and 0.1>c>0.

[0208] Aspect 24. G 1 , G 2 , and Z 2 24. The thermoreversible polymer of any one of aspects 4-23, wherein one or more of is an independently selected modifier selected from heparin, hyaluronic acid, a member of a specific binding pair, a polypeptide, and a nucleic acid.

[0209] Aspect 25. G 1 , G 2 , and Z 2 24. The thermoreversible polymer of any one of embodiments 4-23, wherein one or more of is an independently selected modifier selected from gelatin, fibronectin, collagen, or laminin.

[0210] Aspect 26. G 1 , G 2 and Z 2 24. The thermoreversible polymer of any one of embodiments 4-23, wherein one or more of is a polypeptide selected from a chemokine, a peptide hormone, or a growth factor.

[0211] Embodiment 27. The thermoreversible polymer of embodiment 26, wherein the polypeptide is fibroblast growth factor, epidermal growth factor, hepatic growth factor insulin, stromal cell-derived factor-1, thymosin beta4, sonic hedgehog, noggin, activin, transforming growth factor, bone morphogenetic protein, brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor, neurotrophin 3, platelet-derived growth factor, FGF-2, FGF-8, keratinocyte growth factor, or insulin-like growth factor.

[0212] Embodiment 28. The thermoreversible polymer of embodiment 26, wherein the polypeptide is selected from liver growth factor, bone morphogenetic protein, FGF-2, FGF-8, and keratinocyte growth factor.

[0213] Aspect 29. Formula (IV): Contains TIFF2024521148000097.tif47128, During the ceremony, a, b, c, and d are mole fractions of said comonomers, and a, b, c, and d are each greater than zero; PEG n is a polyethylene glycol polymer, and n is an integer from 1 to 2500; R 1 is alkyl or substituted alkyl; R 2 is alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl; and G 1 and G 2 are each independently selected from a polymer segment, an end group, a linker, and an attached modifying agent; The thermoreversible polymer of any one of embodiments 1 to 3.

[0214] Aspect 30. R 1 is C1-C6 alkyl.

[0215] Aspect 31. R 1 is an alkyl group selected from the group consisting of n-butyl, isobutyl, tert-butyl, n-propyl, pentyl, isopropyl, and isopentyl.

[0216] Aspect 32. R 1 The thermoreversible polymer of embodiment 31, wherein is n-butyl.

[0217] Aspect 33. R 2 The thermoreversible polymer of any one of embodiments 30-32, wherein is an alkoxy group.

[0218] Aspect 34. R 2 is a C1-C6 alkoxy group selected from the group consisting of methoxy, ethoxy, n-propoxy, isoproxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, and isopentoxy.

[0219] Aspect 35. R 2 The thermoreversible polymer of embodiment 34, wherein is methoxy.

[0220] Aspect 36. Formula (V): Contains TIFF2024521148000098.tif59128, During the ceremony, a, b, c, and d are mole fractions of said comonomers, and a, b, c, and d are each greater than zero; PEG nis a polyethylene glycol polymer, and n is an integer from 1 to 2500; R 1 is alkyl or substituted alkyl; R 2 is alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl; and G 1 and G 2 are each independently selected from a polymer segment, an end group, a linker, and an attached modifying agent; The thermoreversible polymer of any one of embodiments 1 to 3.

[0221] Aspect 37. R 1 is C1-C6 alkyl; The thermoreversible polymer of embodiment 36.

[0222] Aspect 38. R 1 38. The thermoreversible polymer of embodiment 37, wherein is an alkyl group selected from the group consisting of n-butyl, isobutyl, tert-butyl, n-propyl, pentyl, isopropyl, and isopentyl.

[0223] Aspect 39. R 1 The thermoreversible polymer of embodiment 38, wherein is n-butyl.

[0224] Aspect 40. R 2 The thermoreversible polymer of any one of embodiments 37-39, wherein is an alkoxy group.

[0225] Aspect 41. R 2 is a C1-C6 alkoxy group selected from the group consisting of methoxy, ethoxy, n-propoxy, isoproxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, and isopentoxy.

[0226] Aspect 42. R 2 The thermoreversible polymer of embodiment 41, wherein is methoxy.

[0227] Aspect 43. Formula (VI): Including TIFF2024521148000099.tif50128, During the ceremony, a, b, c, and d are mole fractions of said comonomers, and a, b, c, and d are each greater than zero; PEG n is a polyethylene glycol polymer, and n is an integer from 1 to 2500; R 1 is alkyl or substituted alkyl; R 2 is alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl; and G 1 and G 2 are each independently selected from a polymer segment, an end group, a linker, and an attached modifying agent; The thermoreversible polymer of any one of embodiments 1 to 3.

[0228] Aspect 44. R 1 is C1-C6 alkyl.

[0229] Aspect 45. R 1 is an alkyl group selected from the group consisting of n-butyl, isobutyl, tert-butyl, n-propyl, pentyl, isopropyl, and isopentyl.

[0230] Aspect 46. R 1 The thermoreversible polymer of embodiment 45, wherein is n-butyl.

[0231] Aspect 47. R 2 The thermoreversible polymer of any one of embodiments 44-46, wherein is an alkoxy group.

[0232] Aspect 48. R 2 is a C1-C6 alkoxy group selected from the group consisting of methoxy, ethoxy, n-propoxy, isoproxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, and isopentoxy.

[0233] Aspect 49. R 2 The thermoreversible polymer of embodiment 48, wherein is methoxy.

[0234] Embodiment 50. Formula (VII): Including TIFF2024521148000100.tif41128, During the ceremony, a, b, c, and d are mole fractions of said comonomers, and a, b, c, and d are each greater than zero; PEG n is a polyethylene glycol polymer, and n is an integer from 1 to 2500; R 1 is alkyl or substituted alkyl; R 2 is alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl; and G 1 and G 2 are each independently selected from a polymer segment, an end group, a linker, and an attached modifying agent; The thermoreversible polymer of any one of embodiments 1 to 3.

[0235] Aspect 51. R 1is C1-C6 alkyl.

[0236] Aspect 52. R 1 is an alkyl group selected from the group consisting of n-butyl, isobutyl, tert-butyl, n-propyl, pentyl, isopropyl, and isopentyl.

[0237] Aspect 53. R 1 53. The thermoreversible polymer of embodiment 52, wherein is n-butyl.

[0238] Aspect 54. R 2 54. The thermoreversible polymer of any one of embodiments 51-53, wherein is an alkoxy group.

[0239] Aspect 55. R 2 is a C1-C6 alkoxy group selected from the group consisting of methoxy, ethoxy, n-propoxy, isoproxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, and isopentoxy.

[0240] Aspect 56. R 2 56. The thermoreversible polymer of embodiment 55, wherein

[0241] Aspect 57. Formula (VIII): Including TIFF2024521148000101.tif40128, During the ceremony, a, b, c, and d are mole fractions of said comonomers, and a, b, c, and d are each greater than zero; PEG n is a polyethylene glycol polymer, and n is an integer from 1 to 2500; R 1 is alkyl or substituted alkyl; R 2is alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl; and G 1 and G 2 are each independently selected from a polymer segment, an end group, a linker, and an attached modifying agent; The thermoreversible polymer of any one of embodiments 1 to 3.

[0242] Aspect 58. R 1 is C1-C6 alkyl.

[0243] Aspect 59. R 1 is an alkyl group selected from the group consisting of n-butyl, isobutyl, tert-butyl, n-propyl, pentyl, isopropyl, and isopentyl.

[0244] Aspect 60. R 1 is n-butyl.

[0245] Aspect 61. R 2 The thermoreversible polymer of any one of embodiments 58-60, wherein is an alkoxy group.

[0246] Aspect 62. R 2 is a C1-C6 alkoxy group selected from the group consisting of methoxy, ethoxy, n-propoxy, isoproxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, and isopentoxy.

[0247] Aspect 63. R 2 The thermoreversible polymer of embodiment 62, wherein

[0248] Aspect 64. Formula (IX): Including TIFF2024521148000102.tif40128, During the ceremony, a, b, c, and d are mole fractions of said comonomers, and a, b, c, and d are each greater than zero; PEG n is a polyethylene glycol polymer, and n is an integer from 1 to 2500; R 1 is alkyl or substituted alkyl; R 2 is alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl; and G 1 and G 2 are each independently selected from a polymer segment, an end group, a linker, and an attached modifying agent; The thermoreversible polymer of any one of embodiments 1 to 3.

[0249] Aspect 65. R 1 is C1-C6 alkyl.

[0250] Aspect 66. R 1 is an alkyl group selected from the group consisting of n-butyl, isobutyl, tert-butyl, n-propyl, pentyl, isopropyl, and isopentyl.

[0251] Aspect 67. R 1 67. The thermoreversible polymer of embodiment 66, wherein is n-butyl.

[0252] 68. R 2 The thermoreversible polymer of any one of embodiments 65-67, wherein is an alkoxy group.

[0253] 69. R2 is a C1-C6 alkoxy group selected from the group consisting of methoxy, ethoxy, n-propoxy, isoproxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, and isopentoxy.

[0254] Aspect 70. R 2 70. The thermoreversible polymer of embodiment 69, wherein is methoxy.

[0255] Aspect 71. Formula (X): Including TIFF2024521148000103.tif47128, During the ceremony, a, b, c, and d are mole fractions of said comonomers, and a, b, c, and d are each greater than zero; PEG n is a polyethylene glycol polymer, and n is an integer from 1 to 2500; R 1 is alkyl or substituted alkyl; R 2 is alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl; and G 1 and G 2 are each independently selected from a polymer segment, an end group, a linker, and an attached modifying agent; The thermoreversible polymer of any one of embodiments 1 to 3.

[0256] Aspect 72. R 1 is C1-C6 alkyl.

[0257] Aspect 73. R 1is an alkyl group selected from the group consisting of n-butyl, isobutyl, tert-butyl, n-propyl, pentyl, isopropyl, and isopentyl.

[0258] Aspect 74. R 1 74. The thermoreversible polymer of embodiment 73, wherein is n-butyl.

[0259] Aspect 75. R 2 The thermoreversible polymer of any one of embodiments 72-74, wherein is an alkoxy group.

[0260] Aspect 76. R 2 is a C1-C6 alkoxy group selected from the group consisting of methoxy, ethoxy, n-propoxy, isoproxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, and isopentoxy.

[0261] Aspect 77. R 2 77. The thermoreversible polymer of embodiment 76, wherein is methoxy.

[0262] Aspect 78. Formula (XI): TIFF2024521148000104.tif59128 included, During the ceremony, a, b, c, and d are mole fractions of said comonomers, and a, b, c, and d are each greater than zero; PEG n is a polyethylene glycol polymer, and n is an integer from 1 to 2500; R 1 is alkyl or substituted alkyl; R 2 is alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl; and G1 and G 2 are each independently selected from a polymer segment, an end group, a linker, and an attached modifying agent; The thermoreversible polymer of any one of embodiments 1 to 3.

[0263] Aspect 79. R 1 is C1-C6 alkyl.

[0264] 80. R 1 80. The thermoreversible polymer of embodiment 79, wherein is an alkyl group selected from the group consisting of n-butyl, isobutyl, tert-butyl, n-propyl, pentyl, isopropyl, and isopentyl.

[0265] 81. R 1 The thermoreversible polymer of embodiment 80, wherein is n-butyl.

[0266] Aspect 82. R 2 The thermoreversible polymer of any one of embodiments 79-81, wherein is an alkoxy group.

[0267] Aspect 83. R 2 is a C1-C6 alkoxy group selected from the group consisting of methoxy, ethoxy, n-propoxy, isoproxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, and isopentoxy.

[0268] 84. R 2 The thermoreversible polymer of embodiment 83, wherein is methoxy.

[0269] Embodiment 85. Formula (XII): TIFF2024521148000105.tif58128 included, During the ceremony, a, b, c, and d are mole fractions of said comonomers, and a, b, c, and d are each greater than zero; PEG nis a polyethylene glycol polymer, and n is an integer from 1 to 2500; R 1 is alkyl or substituted alkyl; R 2 is alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl; and G 1 and G 2 are each independently selected from a polymer segment, an end group, a linker, and an attached modifying agent; The thermoreversible polymer of any one of embodiments 1 to 3.

[0270] Aspect 86. R 1 is C1-C6 alkyl.

[0271] Aspect 87. R 1 87. The thermoreversible polymer of embodiment 86, wherein is an alkyl group selected from the group consisting of n-butyl, isobutyl, tert-butyl, n-propyl, pentyl, isopropyl, and isopentyl.

[0272] 88. R 1 88. The thermoreversible polymer of embodiment 87, wherein is n-butyl.

[0273] 89. R 2 The thermoreversible polymer of any one of embodiments 86-88, wherein is an alkoxy group.

[0274] Aspect 90. R 2 is a C1-C6 alkoxy group selected from the group consisting of methoxy, ethoxy, n-propoxy, isoproxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, and isopentoxy.

[0275] Aspect 91. R 2 The thermoreversible polymer of embodiment 90, wherein is methoxy.

[0276] Aspect 92. Formula (XIII): TIFF2024521148000106.tif42128, During the ceremony, a, b, c, and d are mole fractions of said comonomers, and a, b, c, and d are each greater than zero; PEG n is a polyethylene glycol polymer, and n is an integer from 1 to 2500; R 1 is alkyl or substituted alkyl; R 2 is alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl; and G 1 and G 2 are each independently selected from a polymer segment, an end group, a linker, and an attached modifying agent; The thermoreversible polymer of any one of embodiments 1 to 3.

[0277] Aspect 93. R 1 is C1-C6 alkyl.

[0278] Aspect 94. R 1 94. The thermoreversible polymer of embodiment 93, wherein is an alkyl group selected from the group consisting of n-butyl, isobutyl, tert-butyl, n-propyl, pentyl, isopropyl, and isopentyl.

[0279] Aspect 95. R 1 95. The thermoreversible polymer of embodiment 94, wherein is n-butyl.

[0280] Aspect 96. R 2 The thermoreversible polymer of any one of embodiments 93-95, wherein is an alkoxy group.

[0281] Aspect 97. R 2 is a C1-C6 alkoxy group selected from the group consisting of methoxy, ethoxy, n-propoxy, isoproxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, and isopentoxy.

[0282] Aspect 98. R 2 98. The thermoreversible polymer of embodiment 97, wherein is methoxy.

[0283] Aspect 99. Formula (XIV): Including TIFF2024521148000107.tif40128, During the ceremony, a, b, c, and d are mole fractions of said comonomers, and a, b, c, and d are each greater than zero; PEG n is a polyethylene glycol polymer, and n is an integer from 1 to 2500; R 1 is alkyl or substituted alkyl; R 2 is alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl; and G 1 and G 2 are each independently selected from a polymer segment, an end group, a linker, and an attached modifying agent; The thermoreversible polymer of any one of embodiments 1 to 3.

[0284] Aspect 100. R 1 is C1-C6 alkyl.

[0285] Aspect 101. R 1 is an alkyl group selected from the group consisting of n-butyl, isobutyl, tert-butyl, n-propyl, pentyl, isopropyl, and isopentyl.

[0286] Aspect 102. R 1 The thermoreversible polymer of embodiment 101, wherein is n-butyl.

[0287] Aspect 103. R 2 The thermoreversible polymer of any one of embodiments 100-102, wherein is an alkoxy group.

[0288] Aspect 104. R 2 is a C1-C6 alkoxy group selected from the group consisting of methoxy, ethoxy, n-propoxy, isoproxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, and isopentoxy.

[0289] Aspect 105. R 2 The thermoreversible polymer of embodiment 104, wherein is methoxy.

[0290] Aspect 106. Formula (XV): TIFF2024521148000108.tif40128 included, During the ceremony, a, b, c, and d are mole fractions of said comonomers, and a, b, c, and d are each greater than zero; PEG n is a polyethylene glycol polymer, and n is an integer from 1 to 2500; R 1 is alkyl or substituted alkyl; R 2is alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl; and G 1 and G 2 are each independently selected from a polymer segment, an end group, a linker, and an attached modifying agent; The thermoreversible polymer of any one of embodiments 1 to 3.

[0291] Aspect 107. R 1 is C1-C6 alkyl.

[0292] Aspect 108. R 1 is an alkyl group selected from the group consisting of n-butyl, isobutyl, tert-butyl, n-propyl, pentyl, isopropyl, and isopentyl.

[0293] Aspect 109. R 1 The thermoreversible polymer of embodiment 108, wherein is n-butyl.

[0294] Aspect 110. R 2 The thermoreversible polymer of any one of embodiments 107-109, wherein is an alkoxy group.

[0295] Aspect 111. R 2 is a C1-C6 alkoxy group selected from the group consisting of methoxy, ethoxy, n-propoxy, isoproxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, and isopentoxy.

[0296] Aspect 112. R 2 The thermoreversible polymer of embodiment 111, wherein is methoxy.

[0297] Embodiment 116 The thermoreversible polymer of any one of embodiments 1 to 115, which is solid at or above 20° C.

[0298] Embodiment 117. The thermoreversible polymer of any one of embodiments 1 to 115, which is solid at 37° C.

[0299] Embodiment 118. The thermoreversible polymer of any one of embodiments 1 to 115, which is liquid at or below 30° C.

[0300] Embodiment 119. The thermoreversible polymer of any one of embodiments 1 to 115, which is liquid at 4° C.

[0301] Embodiment 120. A method for making a thermoreversible polymer, comprising: a) copolymerizing N-isopropylacrylamide and N-acryloxysuccinimide to form a first copolymer comprising an acrylic backbone; b) contacting the copolymer with an alkylamine and an alkoxypolyethylene glycol amine to form a second copolymer; and c) contacting the second copolymer with isopropylamine to form a copolymer of Formula I: Generating polymer of TIFF2024521148000109.tif40128 Including, During the ceremony, a, b, and c are mole fractions of comonomers, each of a, b, and c being greater than zero; PEG n is a polyethylene glycol polymer, and n is an integer from 1 to 2500; X is independently selected from C, O, and NH; R 1 is alkyl or substituted alkyl; R 2 is alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl; and G 1 and G 2 are each independently selected from a polymer segment, an end group, a linker, and an attached modifying agent; The above-mentioned production method.

[0302] Aspect 121. R 1 is C1-C6 alkyl.

[0303] Aspect 122. R 1 122. The method of embodiment 121, wherein is an alkyl group selected from the group consisting of n-butyl, isobutyl, tert-butyl, n-propyl, pentyl, isopropyl, and isopentyl.

[0304] Aspect 123. R 1 The method of embodiment 122, wherein is n-butyl.

[0305] Aspect 124. R 2 The method of any one of embodiments 121-123, wherein is an alkoxy group.

[0306] Aspect 125. R 2 is a C1-C6 alkoxy group selected from the group consisting of methoxy, ethoxy, n-propoxy, isoproxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, and isopentoxy.

[0307] Aspect 126. R 2 The method of embodiment 125, wherein is methoxy.

[0308] Embodiment 127. The method of any one of embodiments 120 to 126, wherein a>0.8, 0.1>b>0, and 0.2>c>0.

[0309] Embodiment 128. A method of producing a copolymer comprising the steps of: a) copolymerizing N-isopropylacrylamide and N-acryloxysuccinimide to produce a first copolymer comprising an acrylic backbone; b) contacting the copolymer with butylamine and methoxypolyethylene glycol amine to produce a second copolymer; and c) contacting the second copolymer with isopropylamine to produce a copolymer of Formula II: Generating polymer of TIFF2024521148000110.tif42128 Including, During the ceremony, n is an integer from 1 to 2500, and G 1 and G 2 are each independently selected from a polymer segment, an end group, a linker, and an attached modifying agent; The method of any one of aspects 120 to 127.

[0310] Embodiment 129. A method for making a thermoreversible polymer, comprising: copolymerizing N-isopropylacrylamide, N-acryloxysuccinimide, and an alkyl methacrylate to form a first copolymer comprising an acrylic backbone, contacting the copolymer with an alkoxypolyethylene glycol amine to form a second copolymer, and contacting the second copolymer with isopropylamine to form a copolymer of Formula I: Generating polymer of TIFF2024521148000111.tif40128 Including, During the ceremony, a, b, and c are mole fractions of comonomers, each of a, b, and c being greater than zero; PEG n is a polyethylene glycol polymer, and n is an integer from 1 to 2500; R 1 is alkyl or substituted alkyl; R 2is alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl; and G 1 and G 2 are each independently selected from a polymer segment, an end group, a linker, and an attached modifying agent; The above-mentioned production method.

[0311] Embodiment 130. A method for making a thermoreversible polymer, comprising: copolymerizing N-isopropylacrylamide and N-acryloxysuccinimide to produce a first copolymer comprising an acrylic backbone, contacting the copolymer with an alkylamine (e.g., butylamine) and an alkoxypolyethylene glycol amine to produce a second copolymer, contacting the second copolymer with an aminoalkyl methacrylate (e.g., 2-amino methacrylate) to produce a third copolymer, and contacting the third copolymer with isopropylamine to produce a copolymer of formula IV: Generating polymer of TIFF2024521148000112.tif47128 Including, During the ceremony, a, b, and c are mole fractions of comonomers, each of a, b, and c being greater than zero; PEG n is a polyethylene glycol polymer, and n is an integer from 1 to 2500; R 1 is alkyl or substituted alkyl; R 2is alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl; and G 1 and G 2 are each independently selected from a polymer segment, an end group, a linker, and an attached modifying agent; The above-mentioned production method.

[0312] 131. A method for making a thermoreversible polymer, comprising: Copolymerizing N-isopropylacrylamide and N-acryloxysuccinimide with a RAFT agent (e.g., a DMP RAFT agent) to form a first copolymer comprising an acrylic backbone, contacting the copolymer with an alkylamine and an alkoxypolyethylene glycol amine to form a second copolymer, and contacting the second copolymer with isopropylamine to form a copolymer of Formula I: Generating polymer of TIFF2024521148000113.tif40128 Including, During the ceremony, a, b, and c are mole fractions of comonomers, each of a, b, and c being greater than zero; PEG n is a polyethylene glycol polymer, and n is an integer from 1 to 2500; R 1 is alkyl or substituted alkyl; R 2 is alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl; and G 1 but, TIFF2024521148000114.tif13128, where: TIFF2024521148000115.tif2128 is G 1 and the polymer, G 2 but, TIFF2024521148000116.tif12128, where: TIFF2024521148000117.tif2128 is G 2 and the polymer, The above-mentioned production method.

[0313] Aspect 132. a) a thermoreversible polymer according to any one of embodiments 1 to 119, and b) Buffer aqueous solution 1. A hydrogel composition comprising:

[0314] Embodiment 133. The hydrogel composition of embodiment 132, further comprising cells.

[0315] Embodiment 134. The hydrogel composition of embodiment 133, wherein the cells are stem cells selected from the group consisting of: (a) adult stem cells derived from bone marrow, umbilical cord tissue, or placenta; (b) neural stem cells; (c) progenitor cells derived from embryonic stem cells; and (d) embryonic stem cells.

[0316] Embodiment 135. The hydrogel composition of embodiment 134, wherein the cells are mesenchymal stem cells or hematopoietic stem cells.

[0317] Embodiment 136 The hydrogel composition of embodiment 135, wherein the cell is an immune cell.

[0318] Embodiment 137. The hydrogel composition of embodiment 136, wherein the immune cell is a T cell or a natural killer cell.

[0319] Embodiment 138 The hydrogel composition of any one of embodiments 133 to 137, wherein the cells are genetically modified.

[0320] Embodiment 140 The hydrogel composition of embodiment 137, wherein the cell is a T cell genetically modified to produce a chimeric antigen receptor.

[0321] Aspect 141. A method for expanding cells, comprising: a) introducing cells into the hydrogel composition of aspect 140 to produce a culture mixture; and b) incubating the culture mixture under conditions suitable for the expansion of the cells. EXAMPLES

[0322] The following examples are presented to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors perceive as their invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy of numbers used (e.g., amounts, temperature, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, such as bp, base pairs; kb, kilobases; pl, picoliters; s or sec, seconds; min, minutes; h or hr, hours; aa, amino acids; kb, kilobases; bp, base pairs; nt, nucleotides; im, intramuscular; ip, intraperitoneal; sc, subcutaneous, etc.

[0323] Example 1: Polymer synthesis example We have synthesized fully defined synthetic thermoreversible random copolymers based on the interaction of hydrophilic components (such as poly(ethylene glycol) (PEG)) with temperature-sensitive poly(N-isopropylacrylamide) (PNIPAAm). To generate novel thermoreversible graft copolymers, a two-step synthetic process was developed, where PEG represents the hydrophilic block, PNIPAAm represents the hydrophobic block, and alkyl pendant groups (described as butyl chains in this example, but can be any alkyl chain) act as temperature-shifting moieties (Figure 1). To generate the thermoreversible graft copolymers, a mixture of NIPAAm and N-acryloxysuccinimide (NASI) was first copolymerized by standard radical polymerization. The resulting functionalizable copolymer, after reprecipitation and drying, was mixed with an amine-terminated alkyl group (butylamine in this example) and a monoamine-terminated PEG block. Both amine end groups were attached to the PNIPAAm-co-PNASI backbone via an amidation reaction of the amine with N-hydroxysuccinimide (NHS). Finally, the remaining NHS groups were converted to NIPAAm by adding isopropylamine, and the resulting polymer was dried, dialyzed, and lyophilized. H-NMR characterization of the final polymer (FIG. 2A) indicated the presence of PNIPAAm, PEG, and butylamine. Furthermore, GPC characterization showed a polydispersity index (PDI) of 3 with an obvious lower molecular weight cutoff due to dialysis (FIG. 2B). The final thermoreversible graft copolymer was then reconstituted at the desired weight percentage in a defined cell culture medium for further characterization of the material and cell culture.

[0324] Many general references are available that describe generally known chemical synthetic schemes and conditions useful for synthesizing the disclosed compounds (see, for example, Smith and March, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Fifth Edition, Wiley-Interscience, 2001; or Vogel, A Textbook of Practical Organic Chemistry, Including Qualitative Organic Analysis, Fourth Edition, New York: Longman, 1978).

[0325] The compounds described herein can be purified by any of the means known in the art, including chromatographic means such as high performance liquid chromatography (HPLC), preparative thin layer chromatography, flash column chromatography, and ion exchange chromatography. Any suitable stationary phase can be used, including normal phase and reverse phase, and ionic resin. For example, see Introduction to Modern Liquid Chromatography, 2nd Edition, ed.L.Snyder and J.J.Kirkland, John Wiley and Sons, 1979; and Thin Layer Chromatography, ed.E.Stahl, Springer-Verlag, New York, 1969.

[0326] During any of the processes for preparing the compounds of the present disclosure, it may be necessary and / or desirable to protect sensitive or reactive groups on any of the molecules concerned. This can be achieved by conventional protecting groups, as described in standard works such as TW Greene and PG M Huts, "Protective Groups in Organic Synthesis", Fourth edition, Wiley, New York 2006. The protecting groups can be removed at a convenient subsequent stage using methods known in the art.

[0327] The compounds described herein may contain one or more chiral centers and / or double bonds and therefore can exist as stereoisomers, such as double bond isomers (i.e., geometric isomers), enantiomers, or diastereomers. Thus, all possible enantiomers and stereoisomers of the compounds, including stereomerically pure forms (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure), as well as mixtures of enantiomers and stereoisomers, are included in the description of the compounds herein. Mixtures of enantiomers and stereoisomers can be resolved into their component enantiomers or stereoisomers using separation techniques or chiral synthesis techniques well known to those skilled in the art. The compounds can also exist in several tautomeric forms, including enol forms, keto forms, and mixtures thereof. Thus, the chemical structures depicted herein encompass all possible tautomeric forms of the depicted compounds. The compounds described also include isotopically labeled compounds in which one or more atoms have an atomic mass different from that conventionally found in nature. Examples of isotopes that can be incorporated into the compounds disclosed herein include: 2 H, 3 H, 11 C. 13 C. 14 C. 15 N, 18 O. 17Compounds can exist in unsolvated and solvated forms, including hydrated forms. In general, compounds can be hydrated or solvated. Certain compounds can exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated herein and are intended to be within the scope of the present disclosure.

[0328] The nomenclature used herein to name the subject compounds is set forth in the Examples herein. Where possible, this nomenclature is typically derived using the commercially available AutoNom software (MDL, San Leandro, Calif.).

[0329] Examples of synthesis are shown in FIG. 1 and FIG.

[0330] Figure 1 shows a schematic diagram of the synthesis of the thermoreversible polymer of the present disclosure. Butylamino is shown but can be substituted with another lower alkylamine. The PEG-monoamine is shown as a methoxy group but other functional groups can be used. BAm = butylamine, NIPAAm = N-isopropylacrylamide.

[0331] Figures 2A and 2B show the characterization of the thermoreversible polymer shown in Figure 1. Figure 2A: NMR of poly(NIPAAm-co-BAm)-b-PEG. Figure 2B: Gel permeation chromatography (GPC) of poly(NIPAAm-co-BAm)-b-PEG.

[0332] Figures 3A-3C show data illustrating the mechanical properties of the thermoreversible polymers of Figure 1. Figure 3A: Gelation stiffness vs. temperature for poly(NIPAAm-co-Bam)-b-PEG with varying mol% of BAm. Polymer solutions were tested at 10 wt%. The LCST of the sol-gel transition is shown and defined as the point where G'>G". Figure 3B: Hydrogel stiffness at 37C. Polymer solutions were tested at 10 wt%. Figure 3C: Thermoreversible properties of polymers tested at 10 wt% with 10 mol% BAm.

[0333] Figures 3D-3G show data illustrating the mechanical properties of the thermoreversible polymers of Figure 1. Figure 3D: Thermoreversible properties of the polymer tested at 10 wt% using 10 mol% BAm. Figure 3E: Gelation stiffness vs. temperature of poly(NIPAAm-co-BAm)-b-PEG with varying mol% of alkylamine components such as n-butyl, tert-butyl, or iso-butylamine. The polymer solutions were tested at 10 wt%. The LCST of the sol-gel transition is shown and defined as the point where G'>G". Figure 3F: Effect of mole% of n-butylamine on the LCST of the polymer. Figure 3G: Effect of polymer wt% on the stiffness of the hydrogel obtained in 37C.

[0334] After designing a synthesis strategy for thermoreversible graft copolymers, we investigated key material properties of the hydrogels. These properties include thermoreversible gelation with storage and loss moduli of the polymer at various temperatures (Figure 3D), hydrogel stability, the effect of alkylamine structure on the thermoreversible polymer LCST (Figure 3E), the effect of alkylamine amount on the thermoreversible polymer LCST (Figure 3F), and the effect of polymer weight % on stiffness (Figure 3G). The addition of alkylamines allowed for tight control of the LCST over a range of temperatures. Control of the LCST is important to enable liquid handling and extrusion, while stiffness is important to support and control cell growth and differentiation, long-term stability in aqueous environments, and reproducible synthesis.

[0335] Furthermore, this approach shows the advantage of using mono-conjugated PEG to graft onto the activated PNIPAAm backbone, as it prevents unintended covalent crosslinking that can affect the gel integrity (Figure 4A), and the presence of diamino PEG to conjugate hydrophilic groups allows for covalent crosslinking at higher reaction concentrations. Since this covalent crosslinking is a mix of chemical and physical crosslinking (Figure 4B), it can prevent complete liquefaction of the polymer (Figure 4B) and affect the stiffness of the final hydrogel. Higher reaction yields are obtained by using mono-PEG amine, as the wt% in solvent is not a limiting factor (Figure 4C). Finally, while the use of diamino PEG introduces batch-to-batch variability in the stiffness of the final hydrogel (Figure 4D), the use of mono-PEG amine results in a highly reproducible thermoreversible polymer synthesis.

[0336] Figures 4A-4D show the improved reproducibility and scalability of the thermoreversible polymer of Figure 1 by PEG-monoamine synthesis. Figure 4A: Representative images of undesired covalent crosslinks during polymer synthesis. Representative images of each condition before and after amine / NHS conjugation. PEG diamine was added to poly(NIPAAm-co-NASI) while PEG monoamine was added and reacted in chloroform for 24 hours at room temperature. Figure 4B: Effect of covalent crosslinks on polymer mechanical properties. Figure 4C: Maximum polymer yield determined by reaction concentration without undesired covalent crosslinks. Figure 4D. Effect of mono PEG amine on thermoreversible polymer synthesis.

[0337] Figures 5A-5B show the functionalization of thermoreversible polymers. Figure 5A: Example of a synthetic schematic for introducing chemical functionalization sites into thermoreversible polymers. Figure 5B: Table illustrating possible functionalization molecules / structures and bioconjugation methods of thermoreversible polymers.

[0338] Example 2 : Stem cell differentiation and proliferation Thermohydrogels made with the reversible polymer described in Example 1 were used to grow and differentiate human pluripotent stem cells (hPSCs). hPSCs were induced to differentiate into dopaminergic neurons, cardiomyocytes, or hepatocytes. hPSCs in the hydrogels were cultured in the media described below and marker expression was analyzed on day 25 (for dopaminergic neurons), day 15 (for cardiomyocytes), or day 13 (for hepatocytes). Marker expression was analyzed by immunochemistry and flow cytometry. Results were compared to differentiation of hPSCs in the same media, but in 2D Matrigel.

[0339] Stem cell proliferation Human pluripotent stem cells (hPSCs) were propagated in 3D culture for five passages using thermoreversible hydrogels made with the reversible polymer described in Example 1. hPSCs were seeded as single cells or clusters in the thermoreversible hydrogels and maintained in stem cell culture medium (Essential 8 or mTeSR) in the presence or absence of rock inhibitors (Ri; Y-27632) for 4 days (Figure 6A). Cell aggregates grown in the hydrogels (Figure 6B) were then harvested by cooling / liquefying the hydrogels, and the aggregates were singled and reseeded in the hydrogels to constitute one passage. This process was continued for five consecutive passages in the hydrogels. The expression of pluripotency markers was analyzed by immunochemistry, flow cytometry, and predicted to maintain pluripotency using commercially available PluriTest.

[0340] result hPSCs grown in thermoreversible hydrogels showed stable cell proliferation, with a ~20-fold change in yield after 4 days of expansion over five passages (Figure 6C). After five passages in hydrogels, the resulting hPSCs maintained their pluripotent potential as measured by PluriTest scores (Figure 6D) and pluripotency marker expression compared to standard 2D cultures on Matrigel (Figure 6E).

[0341] Differentiation of dopaminergic neurons For neural induction, hPSCs were grown in basal medium (DMDM / Neurobasal / N2 / B27) containing a combination of LDN, SB, PPA, fibroblast growth factor 8 (FGF8), SHH, and CHIR (where "CHIR" is CHIR99021, 2) "LDN" is LDN-193189, SB is SB431542, and PPA is promorphine or SAG), in hydrogels as described in Example 1, or in 2D Matrigel. For neural specification, the medium was switched to basal medium containing brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), L-ascorbic acid (LAA), TGF-β, dibutyryl cyclic AMP (dbCAMP), and the γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT).

[0342] Marker expression was analyzed on day 25. Markers analyzed were FOXA2, Tuj1, and tyrosine hydroxylase (TH).

[0343] Cardiomyocyte differentiation For cardiac mesoderm induction, hPSCs were grown in basal medium (RPMI / B27 without insulin) containing CHIR followed by IWP2, hydrogel as described in Example 1, or 2D Matrigel. For cardiac specification, the medium was changed to basal medium.

[0344] Marker expression was analyzed on day 15. Markers analyzed were cardiac troponin t (cTnT), α-actinin (α-Act), and myosin light chain 2v (MLC2v).

[0345] Hepatocyte differentiation For hepatocyte induction, hPSCs were grown in basal medium (RPMI / B27) containing activin A and CHIR, in hydrogels as described in Example 1, or in 2D Matrigel.

[0346] To specify foregut endoderm, the medium was changed to basal medium containing bone morphogenetic protein 4 (BMP4) and fibroblast growth factor 2 (FGF2).

[0347] To differentiate into hepatocytes, the method was changed to a basal medium containing hepatocyte growth factor (HGF).

[0348] Marker expression was analyzed on day 13. Markers analyzed were alpha-fetoprotein (AFP) and hepatocyte nuclear factor 4α (HNF4α).

[0349] result The results are shown in Figures 7A-7I. Marker expression of cells differentiated in hydrogel ("3D GCP") or Matrigel ("2D Matrigel") is shown in Figures 7B, 7E, and 7H. The production of dopaminergic neurons, cardiomyocytes, and hepatocytes is shown in Figures 7C, 7F, and 7I, respectively.

[0350] Example 3: Synthesis and Characterization of Graft Copolymer (GCP) Butyl Methacrylate (BMA) In some situations, a less viscous liquid form of the thermoreversible hydrogel may be advantageous for applications such as liquid handling. A variation of the graft copolymer was synthesized using an alkyl methacrylate to shift the temperature instead of a second reaction of butylamine with the free NHS groups of NASI. The ability to control the temperature is important for downstream cell manufacturing applications, and the alkyl methacrylate offers the ability to shift the hydrophobicity of the polymer backbone lower with a smaller amount of temperature shifting moiety. The use of butyl methacrylate in the first stage of the synthesis (Figure 8), followed by PEG grafting, resulted in a thermoreversible polymer with a similar terminal storage modulus (Figure 13A) at a lower polymer weight % in solution compared to the thermoreversible polymer described in Figure 1 (Figure 13B).

[0351] FIG. 8 shows the synthesis of a thermoreversible graft copolymer (GCP) made using butyl methacrylate (BMA) as the temperature shifting moiety (GCP-BMA).

[0352] Figures 13A-C show data showing the mechanical properties of GCP-BMA. Figure 13A: GCP-BMA storage modulus at various weight percents in solution compared to butylamine (BAm) conjugated thermoreversible graft copolymer (GCP-BAm). Figure 13B: Stiffness of GCP-BMA matched with comparable stability to GCP-BAm at lower weight percents. Figure 13C: Comparison of GCP-BMA at various weight percents.

[0353] Example 4: Functionalization of Thermoreversible Polymers Bioconjugation of peptides, proteins, and other proteoglycans is an important application for cell growth and differentiation. The ability to present or sequester growth factors, cell-binding peptides, or chemical regulators of signaling pathways provides control of the microenvironment that can affect cell fate. Six independent motifs were devised to provide bioconjugation sites and schematics for thermoreversible polymers. These motifs allow for direct bioconjugation to the polymer backbone.

[0354] 9A-9C and 11A-11D show schematics of functionalization of GCPs. Free methacrylate, thiol, alkyne, carboxylic acid, maleimide, and strained alkyne groups were covalently attached to the polymer backbone reactants of linking amines to activated NHS esters present in the backbone prior to saturation. Each group can be utilized as a handle to directly append proteins, peptides, hyaluronic acid, biotin / streptavidin, heparin, etc. to the polymer backbone.

[0355] 10A-10D show rheometric analysis of functionalized GCPs. The presence of 2 mol% functional groups appears to affect the loss modulus but does not affect the structure or stability of the final gel.

[0356] Example 5: Synthesis of Thermoreversible Polymers Using RAFT A typical radical polymerization with an initiator using AIBN produces polymers of various polydispersity indexes. This distribution of polymer lengths can affect the final mechanical properties of the thermoreversible polymer, including stiffness, LCST, and viscosity. Radical addition-fragmentation chain transfer (RAFT) is a type of living polymerization technique that allows for a more controlled addition of monomers to the growing polymer, resulting in monodisperse polymer lengths. To control the molecular weight of the thermoreversible polymer, graft copolymers were synthesized in the presence of the RAFT agent 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid (DMP). This chain transfer agent controls the addition of monomers to the growing chains and the molecular weight of the resulting polymer when the monomers are present and in a defined ratio relative to the AIBN initiator. Additionally, the remaining chain transfer reagent can be reduced to a free thiol for use in bioconjugation.

[0357] FIG. 12 shows a schematic of the GCP-RAFT synthesis.

[0358] Although the present invention has been described with reference to specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process steps or steps to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

1. N-isopropylacrylamide (NIPAM) comonomer, a lower alkylamine comonomer, and Poly(ethylene glycol) (PEG) comonomer A thermoreversible polymer comprising: The thermoreversible polymer, wherein the terminal PEG monomer is substituted with alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl.

2. the lower alkyl amine comonomer comprises n-butyl, isobutyl, tert-butyl, n-propyl, pentyl, isopropyl, or isopentyl; and the terminal PEG monomer is substituted with an alkoxy group; The thermoreversible polymer of claim 1.

3. 3. The thermoreversible polymer of claim 2, wherein the alkoxy group is a C1-C6 alkoxy selected from the group consisting of methoxy, ethoxy, n-propoxy, isoproxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, and isopentoxy.

4. Formula (I): Including, During the ceremony, a, b, and c are mole fractions of said comonomers, and a, b, and c are each greater than zero; PEG ｎ is a polyethylene glycol polymer; n is an integer from 1 to 2500; R １ is alkyl or substituted alkyl; R ２ is alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl; and G １ and G ２ are each independently selected from a polymer segment, an end group, a linker, and an attached modifying agent; The thermoreversible polymer of claim 1.

5. R １ The thermoreversible polymer of claim 4, wherein is a C1-C6 alkyl.

6. R １ 6. The thermoreversible polymer of claim 5, wherein is an alkyl group selected from the group consisting of n-butyl, isobutyl, tert-butyl, n-propyl, pentyl, isopropyl, and isopentyl.

7. R １ The thermoreversible polymer of claim 6, wherein is n-butyl.

8. R ２ The thermoreversible polymer of claim 4 , wherein is an alkoxy group.

9. R ２ 9. The thermoreversible polymer of claim 8, wherein is a C1 to C6 alkoxy group selected from the group consisting of methoxy, ethoxy, n-propoxy, isoproxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, and isopentoxy.

10. R ２ The thermoreversible polymer of claim 9 , wherein is methoxy.

11. 5. The thermoreversible polymer according to claim 4, wherein a>0.8, 0.2>b>0, and 0.1>c>0.

12. Formula II: Including, During the ceremony, n is 1 to 25, and G １ and G ２ are each independently selected from a polymer segment, an end group, a linker, and an attached modifying agent; The thermoreversible polymer of claim 1.

13. The PEG or PEG ｎ 2. The thermoreversible polymer of claim 1, wherein said polymer has a molecular weight of 2 kDa to 100 kDa.

14. Formula (III): Including, During the ceremony, a, b, c, and d are mole fractions of said comonomers, and a, b, c, and d are each greater than zero; PEG ｎ is a polyethylene glycol polymer; n is an integer from 1 to 2500; R １ is alkyl or substituted alkyl; R ２ is alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl; L is a linker, Z ２ is a modifier, and G １ and G ２ are each independently selected from a polymer segment, an end group, a linker, and an attached modifying agent; The thermoreversible polymer of claim 1.

15. R １ The thermoreversible polymer of claim 14, wherein is a C1 to C6 alkyl.

16. R １ 16. The thermoreversible polymer of claim 15, wherein is an alkyl group selected from the group consisting of n-butyl, isobutyl, tert-butyl, n-propyl, pentyl, isopropyl, and isopentyl.

17. R １ The thermoreversible polymer of claim 16, wherein is n-butyl.

18. R ２ The thermoreversible polymer of claim 14 , wherein is an alkoxy group.

19. R ２ 19. The thermoreversible polymer of claim 18, wherein is a C1 to C6 alkoxy group selected from the group consisting of methoxy, ethoxy, n-propoxy, isoproxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, and isopentoxy.

20. R ２ 20. The thermoreversible polymer of claim 19, wherein is methoxy.

21. Z ２ 15. The thermoreversible polymer of claim 14, wherein is a chemoselective functional group selected from thiols, alkynes, cyclooctynes, azides, phosphines, maleimides, alkoxyamines, aldehydes, and protected forms or precursors thereof.

22. Z ２ 15. The thermoreversible polymer of claim 14, wherein is a modifier selected from heparin, hyaluronic acid, a specific binding member, a peptide, a nucleic acid, gelatin, fibronectin, collagen, laminin, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin, progesterone, glucose, stromal cell-derived factor-1 (SDF-1), thymosin beta4, sonic hedgehog (SHH), noggin, activin, transforming growth factor beta (TGF-β) (TGFβ3), FGF8, brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), neurotrophic factor 3 (NT3), platelet-derived growth factor (PDGF), IL-16, IL-2, and insulin-like growth factor 1 (IGF-1).

23. 13. The thermoreversible polymer of claim 12, wherein a>0.8, 0.2>b>0, and 0.1>c>0.

24. G １ , G ２ , and Z ２ 15. The thermoreversible polymer of claim 14, wherein one or more of the following is an independently selected modifier selected from heparin, hyaluronic acid, a member of a specific binding pair, a polypeptide, and a nucleic acid.

25. G １ , G ２ , and Z ２ 15. The thermoreversible polymer of claim 14, wherein one or more of is an independently selected modifier selected from gelatin, fibronectin, collagen, or laminin.

26. G １ , G ２ , and Z ２ 15. The thermoreversible polymer of claim 14, wherein one or more of is a polypeptide selected from a chemokine, a peptide hormone, or a growth factor.

27. 27. The thermoreversible polymer of claim 26, wherein the polypeptide is fibroblast growth factor, epidermal growth factor, hepatic growth factor insulin, stromal cell-derived factor-1, thymosin beta4, sonic hedgehog, noggin, activin, transforming growth factor, bone morphogenetic protein, brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor, neurotrophin 3, platelet-derived growth factor, FGF-2, FGF-8, keratinocyte growth factor, or insulin-like growth factor.

28. 27. The thermoreversible polymer of claim 26, wherein the polypeptide is selected from hepatic growth factor, bone morphogenetic protein, FGF-2, FGF-8, and keratinocyte growth factor.

29. 10. The thermoreversible polymer of claim 1 which is solid at or above 20°C.

30. 10. The thermoreversible polymer of claim 1 which is solid at 37°C.

31. 2. The thermoreversible polymer of claim 1 which is liquid at or below 30°C.

32. 2. The thermoreversible polymer of claim 1 which is liquid at 4°C.

33. 1. A method for making a thermoreversible polymer, comprising: copolymerizing N-isopropylacrylamide and N-acryloxysuccinimide to form a first copolymer comprising an acrylic backbone; contacting the copolymer with an alkyl amine and an alkoxy polyethylene glycol amine to form a second copolymer; and The second copolymer is contacted with isopropylamine to form a copolymer of Formula I: To produce a polymer of Including, During the ceremony, a, b, and c are mole fractions of comonomers, each of a, b, and c being greater than zero; PEG ｎ is a polyethylene glycol polymer; n is an integer from 1 to 2500; R １ is alkyl or substituted alkyl; R ２ is alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl; and G １ and G ２ are each independently selected from a polymer segment, an end group, a linker, and an attached modifying agent; The above-mentioned production method.

34. R １ The method of claim 33, wherein is C1 to C6 alkyl.

35. R １ 35. The method of claim 34, wherein is an alkyl group selected from the group consisting of n-butyl, isobutyl, tert-butyl, n-propyl, pentyl, isopropyl, and isopentyl.

36. R １ The method of claim 34, wherein is n-butyl.

37. R ２ The method of claim 33, wherein is an alkoxy group.

38. R ２ 38. The method of claim 37, wherein is a C1 to C6 alkoxy group selected from the group consisting of methoxy, ethoxy, n-propoxy, isoproxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, and isopentoxy.

39. R ２ The method of claim 38, wherein is methoxy.

40. 34. The method of claim 33, wherein a>0.8, 0.1>b>0, and 0.2>c>0.

41. copolymerizing N-isopropylacrylamide and N-acryloxysuccinimide to form a first copolymer comprising an acrylic backbone; contacting the copolymer with butylamine and methoxypolyethylene glycol amine to produce a second copolymer; and The second copolymer is contacted with isopropylamine to form a copolymer of Formula II: To produce a polymer of Including, During the ceremony, n is an integer from 1 to 2500; and G １ and G ２ are each independently selected from a polymer segment, an end group, a linker, and an attached modifying agent; 34. The method of claim 33.

42. a) a thermoreversible polymer according to any one of claims 1 to 32, and b) Buffer aqueous solution 1. A hydrogel composition comprising:

43. 43. The hydrogel composition of claim 42, further comprising cells.

44. 44. The hydrogel composition of claim 43, wherein the cells are stem cells selected from the group consisting of: (a) adult stem cells derived from bone marrow, umbilical cord tissue, or placenta; (b) neural stem cells; (c) progenitor cells derived from embryonic stem cells; and (d) embryonic stem cells.

45. 45. The hydrogel composition of claim 44, wherein the cells are mesenchymal stem cells or hematopoietic stem cells.

46. 44. The hydrogel composition of claim 43, wherein the cell is an immune cell.

47. 47. The hydrogel composition of claim 46, wherein the immune cell is a T cell or a natural killer cell.

48. 44. The hydrogel composition of claim 43, wherein the cells are genetically modified.

49. 48. The hydrogel composition of claim 47, wherein the cell is a T cell genetically modified to produce a chimeric antigen receptor.

50. A method for expanding cells, comprising the steps of: Introducing cells into the hydrogel composition of claim 42 to produce a culture mixture; and incubating said culture mixture under conditions suitable for growth of said cells; The proliferation method comprising the steps of:

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