[go: up one dir, main page]

WO2025080682A1 - Appareil de plaque de puits - Google Patents

Appareil de plaque de puits Download PDF

Info

Publication number
WO2025080682A1
WO2025080682A1 PCT/US2024/050534 US2024050534W WO2025080682A1 WO 2025080682 A1 WO2025080682 A1 WO 2025080682A1 US 2024050534 W US2024050534 W US 2024050534W WO 2025080682 A1 WO2025080682 A1 WO 2025080682A1
Authority
WO
WIPO (PCT)
Prior art keywords
hydrogel
hydrogels
wells
backbone polymer
well
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2024/050534
Other languages
English (en)
Inventor
Steven R. Caliari
Mackenzie SKELTON
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
UVA Licensing and Ventures Group
Original Assignee
University of Virginia Patent Foundation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Virginia Patent Foundation filed Critical University of Virginia Patent Foundation
Publication of WO2025080682A1 publication Critical patent/WO2025080682A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
    • C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
    • C08J3/075—Macromolecular gels
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/24—Crosslinking, e.g. vulcanising, of macromolecules
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/12—Well or multiwell plates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/14—Scaffolds; Matrices
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
    • C08B37/0012—Cyclodextrin [CD], e.g. cycle with 6 units (alpha), with 7 units (beta) and with 8 units (gamma), large-ring cyclodextrin or cycloamylose with 9 units or more; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2305/00—Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2301/00 or C08J2303/00
    • C08J2305/04—Alginic acid; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2305/00—Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2301/00 or C08J2303/00
    • C08J2305/08—Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2371/00—Characterised by the use of polyethers obtained by reactions forming an ether link in the main chain; Derivatives of such polymers
    • C08J2371/02—Polyalkylene oxides
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2393/00—Characterised by the use of natural resins; Derivatives thereof

Definitions

  • the disclosure in one aspect, relates to an apparatus comprising: a plate; a plurality of wells set into the plate, wherein each individual well of the plurality of wells extends from an outer surface of the plate to an inner surface of the individual well; and a plurality of hydrogels, wherein at least two individual wells of the plurality of wells each comprise a singular hydrogel of the plurality of hydrogels; wherein each individual hydrogel of the plurality of hydrogels comprises a dithiol crosslinker and a first backbone polymer functionalized with an alkene; wherein each individual hydrogel of the plurality of hydrogels comprises a dithiol crosslinkeralkene molar ratio selected from about 1 : 100 to about 1 :1 ; wherein each individual hydrogel of the plurality of hydrogels comprises a weight percentage of the first backbone polymer selected from about 1 % to about 15% of the hydrogel;
  • FIG. 1 A shows a representative illustration of a 6-well plate.
  • FIGS. 1 B-1C illustrate two representative modes of crosslinking that were utilized herein - covalent (FIG. 1 B) and supramolecular (FIG. 1C) - both through the modification of HA.
  • FIG. 1 D shows a representative schematic illustrating how components of the elastic or viscoelastic hydrogel networks come together, where hydrogels can also be modified with thiolated RGD peptide to enable culture of adhesive cells.
  • FIG. 2A shows representative comparisons of stiffness in norHA hydrogels of varying polymer content. Hydrogels in each group had a thiol:norbornene ratio of 0.35.
  • FIG. 2B shows representative comparisons of stiffness in norHA hydrogels with polymer content of 4 wt% and 6 wt% and a changing thiol:norbornene ratio.
  • FIGS. 2E-2F show representative plots of storage and loss moduli at varying frequencies of stiffness-matched viscoelastic (FIG. 2E) and elastic (FIG. 2F) norHA hydrogels.
  • FIG. 3A shows representative plots of fabrication time required per replicate in either a coverslip or a 96-well plate fabrication format.
  • FIG. 3B shows representative plots of the amount of material needed per hydrogel for either a coverslip or a 96-well plate fabrication format.
  • the step size between test points on the coverslip hydrogels and 96-well hydrogels was 1000 pm and 400 pm, respectively.
  • FIG. 7B shows a representative plot of encapsulation viability of NIH3T3 cells encapsulated in viscoelastic or elastic hydrogels after 2 days of culture.
  • FIG. 8 shows a representative 1 H NMR spectrum of a TBA salt of HA (HA-TBA).
  • FIG. 11 shows a representative 1 H NMR spectrum of CD-HA.
  • FIG. 14 shows a representative schematic of the 3D-printed alignment piece used for 96-well hydrogel plate assembly, including its dimensions. All measurements are shown in millimeters.
  • FIG. 15 shows a representative schematic of the 3D-printed mold used for fabrication of the PDMS spacer sheet, including its dimensions. All measurements are shown in millimeters.
  • FIG. 16 shows a representative image of the 96-well plate hydrogel platform used for the leak test, with four hydrogel wells (indicated with arrows) filled with red food coloring. The image was taken 24 hours after adding the food coloring.
  • the term “and/or” includes any and all combinations of one or more of the associated listed items. Expressions such as “at least one of,” when preceding a list of elements, modify the entire list of elements and do not modify the individual elements of the list.
  • references to "a" chemical compound refers to one or more molecules of the chemical compound rather than being limited to a single molecule of the chemical compound. Furthermore, the one or more molecules may or may not be identical, so long as they fall under the category of the chemical compound. Thus, for example, "a" chemical compound is interpreted to include one or more molecules of the chemical, where the molecules may or may not be identical (e.g., different isotopic ratios, enantiomers, and the like).
  • references to "a/an" chemical compound, protein, and antibody each refers to one or more molecules of the chemical compound, protein, and antibody rather than being limited to a single molecule of the chemical compound, protein, and antibody. Furthermore, the one or more molecules may or may not be identical, so long as they fall under the category of the chemical compound, protein, and antibody.
  • ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.
  • a further aspect includes from the one particular value and/or to the other particular value.
  • ranges excluding either or both of those included limits are also included in the disclosure, e g. the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than x’ and less than ‘y’.
  • the range can also be expressed as an upper limit, e.g. ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘less than x’, less than y’, and ‘less than z’.
  • hydrogel generally refers to a network of polymeric material dispersed in a carrier, such as water.
  • amino acid is used interchangeably with “amino acid residue” and may refer to a free amino acid and to an amino acid residue of a peptide. It will be apparent from the context in which the term is used whether it refers to a free amino acid or a residue of a peptide.
  • Synthetic peptides refers a non-naturally occurring peptide. Synthetic peptides can be synthesized, for example, using an automated polypeptide synthesizer. Various solid phase peptide synthesis methods are known in the art.
  • the “degree of modification” of a polymer refers to the stoichiometric ratio between the number of monomer units of the polymer containing a covalently-bonded functional group and the total number of monomer units of the polymer.
  • the monomer unit used for this calculation is the repeating unit of the polymer.
  • the monomer unit of hyaluronic acid would be the disaccharide of D-glucuronic acid and N-acetyl- D-glucosamine, which has a structure represented by the formula:
  • an apparatus comprising: a plate; a plurality of wells set into the plate, wherein each individual well of the plurality of wells extends from an outer surface of the plate to an inner surface of the individual well; and a plurality of hydrogels, wherein at least two individual wells of the plurality of wells each comprise a singular hydrogel of the plurality of hydrogels; wherein each individual hydrogel of the plurality of hydrogels comprises a dithiol crosslinker and a first backbone polymer functionalized with an alkene; wherein each individual hydrogel of the plurality of hydrogels comprises a dithiol crosslinkeralkene molar ratio selected from about 1 : 100 to about 1 :1 ; wherein each individual hydrogel of the plurality of hydrogels comprises a weight percentage of the first backbone polymer selected from about 1% to about 15% of the hydrogel; and wherein at least one hydrogel of the plurality of hydrogels is different from the remaining hydrogels of the pluralit
  • the plurality of hydrogels can be contained within the plurality of wells.
  • an inner surface of a well comprises the individual hydrogel.
  • a first hydrogel and a second hydrogel of the plurality of hydrogels can be different from the remaining hydrogels of the plurality of hydrogels, and the first hydrogel and the second hydrogel are the same as each other.
  • the dithiol crosslinker:alkene molar ratio, the weight percentage of the first backbone polymer, or a combination thereof in at least one hydrogel of the plurality of hydrogels can differ from that of at least one other hydrogel of the plurality of hydrogels.
  • the plurality of wells can include 6, 12, 24, 48, 96, 384, or 1536 wells.
  • the plurality of wells can be arranged in a 2:3 rectangular matrix within the plate.
  • At least two of the wells in the plate can include a hydrogel.
  • two, a third, half, two-thirds, or all of the wells in the plate can comprise a hydrogel, where at least one hydrogel differs from at least one other hydrogel present in the plate.
  • groups of wells can comprise a hydrogel composition, such as columns or rows of wells.
  • the wells in R1 comprise a first hydrogel composition while the wells in R2 comprise a second hydrogel composition that differs from the first hydrogel composition in at least one aspect (e.g., dithiol crosslinkeralkene molar ratio and weight percentage of the first backbone polymer).
  • the well in C1 comprise a first hydrogel composition while the wells in one of the other columns, say C2, comprises a second hydrogel composition that differs from the first hydrogel composition in at least one aspect.
  • the third column C3 can remain empty or comprise a third hydrogel composition, which can be the same as either the first or the second hydrogel composition or be different from either one.
  • the degree of medication of the first backbone polymer can also vary.
  • the first backbone polymer has a degree of modification of about 1 % to about 90%, about 1 % to about 80%, about 1 % to about 70%, about 1 % to about 60%, about 1% to about 50%, about 1 % to about 40%, about 1 % to about 30%, about 5% to about 80%, about 5% to about 60%, about 5% to about 40%, about 10% to about 80%, about 10% to about 60%, about 10% to about 40%, about 5% to about 30%, about 5% to about 25%, about 5% to about 20%, about 10% to about 25%, about 10% to about 20%, or about 15% to about 20%.
  • the degree of modification of the second backbone polymer when functionalized, can also vary.
  • the second backbone polymer has a degree of modification of about 1% to about 90%, about 1 % to about 80%, about 1% to about 70%, about 1% to about 60%, about 1% to about 50%, about 1% to about 40%, about 5% to about 80%, about 5% to about 60%, about 5% to about 40%, about 10% to about 80%, about 10% to about 60%, about 10% to about 40%, about 10% to about 40%, about 15% to about 40%, about 20% to about 40%, about 10% to about 35%, about 10% to about 30%, about 15% to about 40%, about 15% to about 35%, or about 15% to about 30%.
  • the base can be a substrate such as a glass substrate.
  • the base can be functionalized with free thiols, amines, methacrylates, or a combination thereof.
  • the spacer layer can be from about 0.1 mm thick to about 5 mm thick, about 0.5 mm thick to about 5 mm thick, about 0.5 mm thick to no greater than 1 mm thick, 1 mm thick to about 3 mm thick, no greater than 0.5 mm thick, or no less than 1 mm thick to about 2 mm thick.
  • the spacer layer can be made of a material such as silicone.
  • the spacer layer can have the same width and length dimensions as the base.
  • the spacer layer can be set onto the base or fixed by methods such as clamping or fitting the base and spacer into an open box such that they remain aligned with one another.
  • the spacer layer can comprise a plurality of openings each individually having a shape characterized as circular or polygonal.
  • the plurality of openings on the spacer sheet can each have a diameter that is equal to or less than the diameter of an individual well of the plurality of wells within the well-plate.
  • the bottomless well-plate can comprise 6, 12, 24, 48, 96, 384, or 1536 wells.
  • the number of openings in the spacer layer can be equal to or less than the number of wells in the bottomless well-plate.
  • the bottomless well-plate has the same width and length dimensions as the base.
  • the bottomless well-plate can be fixed to the base by means such as an adhesive, clamping, or the like.
  • the hydrogel precursor solution can be any hydrogel or hydrogel composition as disclosed herein.
  • the hydrogel precursor solution can be applied to the base via the openings in the spacer sheet.
  • the solution can be applied manually, such as by pipetting individual hydrogel precursor solutions one at a time per opening in the sheet.
  • the hydrogel precursor solution can be applied to the base using automated or semi-automated means, such as machines configured to simultaneously deliver multiple samples at a time to the base or a robot that can be configured to deliver single or multiple samples to a base with varying degrees of variation between each sample delivered.
  • the base with hydrogel precursor solution applied can be cured using UV radiation, visible light radiation, gamma radiation, electron-beam radiation, or a combination thereof. Curing with UV or visible light radiation can be done at a range of wavelengths, e.g., 320 nm to 640 nm.
  • the hydrogel precursors can be cured via photopolymerization in a UV box in the presence of a photoinitiator. Examples of suitable photoinitiators include, but are not limited to, lithium acylphosphinate (LAP), Irgacure 2959, and Eosin Y.
  • the photopolymerization process can be performed for about 1 minute to about 5 minutes.
  • Aspect 2 The apparatus of aspect 1 , wherein at least a first hydrogel and a second hydrogel of the plurality of hydrogels are different from the remaining hydrogels of the plurality of hydrogels, and wherein the first hydrogel and the second hydrogel are the same as each other.
  • Aspect 7 The apparatus of any one of aspects 1 -4, where the first backbone polymer has a degree of modification of about 1% to about 30%.
  • Aspect 10 The apparatus of aspect 9, wherein the dithiol matrix metalloproteinase peptide comprises the amino acid sequence GCKGGPQGIWGQGKCG (SEQ ID NO:1).
  • Aspect 11 The apparatus of any one of aspects 1-10, wherein each individual hydrogel of the plurality of hydrogels comprises a dithiol crosslinkeralkene molar ratio selected from about 1 :50 to about 1 :1.
  • each individual hydrogel of the plurality of hydrogels comprises a dithiol crosslinkeralkene molar ratio selected from about 1 :25 to about 1 :1.
  • Aspect 14 The apparatus of any one of aspects 1-13, wherein each individual hydrogel of the plurality of hydrogels comprises a weight percentage of the first backbone polymer selected from about 1% to about 10% of the individual hydrogel.
  • Aspect 18 The apparatus of aspect 17, where the second backbone polymer has a degree of modification of about 1% to about 90%.
  • Aspect 22 The apparatus of any one of aspect 16-21 , wherein the viscoelastic hydrogel further comprises a thiolated peptide comprising the amino acid sequence GCKKK (SEQ ID NO:2).
  • Aspect 26 The apparatus of aspect 22 or aspect 23, wherein the viscoelastic hydrogel further comprises an adamantane-p-cyclodextrin-thiolated peptide conjugate and a second backbone polymer.
  • Aspect 27 The apparatus of aspect 22 or aspect 23, wherein the viscoelastic hydrogel further comprises a thiolated peptide conjugate and a second backbone polymer functionalized with adamantane and p-cyclodextrin
  • Aspect 30 The apparatus of any one of aspects 22-29, wherein the at least two hydrogels of the plurality of hydrogels are viscoelastic hydrogels and wherein the backbone functional group:thiolated peptide molar ratio of at least one viscoelastic hydrogel of the plurality of hydrogels differs from that of at least one other viscoelastic hydrogel of the plurality of hydrogels.
  • Aspect 31 The apparatus of any one of aspects 15-30, wherein at least two hydrogels of the plurality of hydrogels are viscoelastic hydrogels.
  • Aspect 32 The apparatus of any one of aspects 1-31 , wherein at least one hydrogel of the plurality of hydrogels further comprises an arginyl-glycyl-aspartic acid (RGD) peptide.
  • RGD arginyl-glycyl-aspartic acid
  • Aspect 33 The apparatus of aspect 32, wherein the RGD peptide comprises the amino acid sequence GCGTGRGDSPG (SEQ ID NO:3).
  • Aspect 35 The apparatus of any one of aspects 1-33, wherein at least half of the plurality of wells each comprise a singular hydrogel of the plurality of hydrogels.
  • Aspect 38 The apparatus of any one of aspects 1-37, wherein the plurality of wells are arranged in a 2:3 rectangular matrix within the plate.
  • a method for fabricating the apparatus of aspect 1 comprising: providing a base; fixing a spacer layer to the base, wherein the spacer layer has a thickness of at least about 0.1 mm and comprises a plurality of openings disposed in the spacer layer; applying at least one hydrogel precursor solution to the base via at least one of the plurality of openings; curing the hydrogel precursor solution with UV light; removing the spacer layer; and fixing a bottomless well-plate to the base, wherein the bottomless well-plate comprises at least six wells; wherein the at least one hydrogel precursor solution comprises the dithiol crosslinker and the first backbone polymer functionalized with an alkene.
  • Aspect 48 The method of any one of aspects 40-47, wherein the hydrogel precursor solution is cured using UV radiation, visible light radiation, gamma radiation, electron-beam radiation, or a combination thereof.
  • Hydrogels can be used to modeling extracellular environments and probe cellular behavior in vitro due to their ability to mimic native tissue properties.
  • In vitro hydrogel models allow for direct, orthogonal control over aspects of the extracellular matrix, such as stiffness 1 - 3 , viscoelasticity 45 , cell adhesion motifs 6 , and presentation of biochemical cues 7 8 .
  • control over viscoelasticity has been shown to dictate stem cell differentiation, with faster relaxing hydrogels promoting preferential osteogenesis 4 .
  • a primary limitation of hydrogel models is their low fabrication throughput, as these platforms are often formed individually on coverslips 9 and cultured within large well plates or dishes.
  • hydrogels can be tuned to modulate stiffness and viscoelasticity for both 2D and 3D cell culture.
  • NorHA is covalently crosslinked with DTT through a light-mediated thiol-ene click reaction to form elastic hydrogels.
  • Viscoelastic hydrogels additionally incorporate p-cyclodextrin-adamantane host-guest interactions, where a thiolated adamantane peptide is tethered to the NorHA through a thiol-ene click reaction.
  • This modular fabrication approach is adaptable to a range of hydrogel mechanics and components such as matrix metalloproteinase (MMP)-degradable crosslinkers and bioactive moieties, while maintaining accessibility to many new users.
  • MMP matrix metalloproteinase
  • these culture platforms are compatible with high-throughput measurement techniques such as microplate readers and high-content imaging systems.
  • hydrogel array was fabricated where HA polymer content and crosslink density were varied to achieve a range of mechanical properties. Following fabrication of the array, hydrogels were swollen overnight in PBS, and the resultant mechanical properties were evaluated using nanoindentation.
  • the in situ characterization of hydrogels in the array is advantageous, as hydrogel mechanics are known to vary with specimen geometry 27 .
  • HA hydrogels of 0.5 wt% ranging up to 4 wt% can span elastic moduli of two orders of magnitude, while keeping the fraction of norbornenes crosslinked the same (FIG. 2A).
  • the solution was vacuum filtered to remove side products from the BOP coupling and dialyzed again in DI water for 5 days before being frozen and lyophilized for long-term storage at -20 °C.
  • CD-HA p-cyclodextrin-modified hyaluronic acid
  • CD-HDA p-cyclodextrin hexamethylene diamine
  • TosCI p-Toluenesulfonyl chloride
  • CD-HDA was reacted with HA-TBA and BOP in anhydrous DMSO at 25°C for 3 h. This reaction was quenched with cold water, dialyzed for 5 days, filtered, and dialyzed for 5 more days. The purified solution was frozen and lyophilized.
  • the degree of modification of HA was determined by 1 H NMR to be 25%, as indicated by integration of hexane linker peaks in FIG. 11 at 0 - 1 .23-1 .68 ppm (12H, ‘a’) relative to the N- acetyl group of HA (3H, 'b’).
  • 96-well Plate Fabrication Glass pieces with the same thickness as a number 1.5 coverslip (0.17 mm, Schott D263) were cut to the dimensions of a 96-well plate (110 x 75 mm, S.l. Howard Glass, Worcester, MA). Bottomless 96-well plates without adhesive (Greiner Bio- One, Kremsmunster, Austria) were bound to microfluidic-grade double-sided adhesive (ARcare 90196NB, Adhesive Research, Glen Rock, PA) that was laser cut to the plate dimensions (FIG. 12). All measurements are shown in millimeters. When adhesive was applied to the bottomless 96-well plates and subsequently the glass bottom, special care was taken to ensure complete adhesion through application of gentle pressure evenly across the plate.
  • silicone spacer sheets of 0.5 mm thickness were laser cut to the 96-well geometry with a diameter of 0.5 mm less than the wells to enable some margin for improper alignment (FIG. 13).
  • a 2 mm-thick polydimethylsiloxane (PDMS) sheet of otherwise same geometry was fabricated.
  • An alignment piece was 3D-printed to assist with alignment of adhesive to plates, silicone sheet to glass, and bottomless plate to glass (FIG. 14).
  • PDMS Spacer Fabrication A mold was 3D printed to assist in the fabrication of 1 mm thick PDMS sheets used to fabricate hydrogels for 3D culture within the 96-well array (FIG. 15). The mold was printed on an Ender-3 S1 3D printer using a 1.75 mm polylactic acid filament (SUNLU, Guangdong, China) at a resolution of 0.2 mm layer height and 30% infill. PDMS was prepared by mixing base and curing agent at a weight ratio of 10:1. The mixture was stirred vigorously and then degassed under vacuum for 1 h. The PDMS mixture was then slowly poured into the mold to avoid the introduction of air bubbles.
  • SUNLU 1.75 mm polylactic acid filament
  • a clear plastic sheet was placed on top of the uncured PDMS and flattened to be flush with the mold to ensure a flat surface.
  • the mold was then placed in an oven at 35°C for 2 h or until the PDMS was fully cured.
  • a 6 mm biopsy punch was used to cut wells out of the PDMS at the site of the mold indentations, and the sheet was cleaned thoroughly before using for 3D hydrogel fabrication.
  • Distinct hydrogel precursor solutions covering the range of mechanical properties described above were mixed with 18 pL of solution pipetted per opening of the silicone spacer sheet fitted onto the thiol-functionalized glass piece of a single 96-well plate.
  • Hydrogel precursors were flattened with a glass piece treated with the hydrophobic coating SigmaCote and photopolymerized (365 nm, 5mW/cm 2 ) in a UV box (VWR) in the presence of 1 mM lithium acylphosphinate (LAP) photoinitiator for 2 min.
  • VWR UV box
  • LAP lithium acylphosphinate
  • EdU Proliferation Assay Proliferative activity of 2D hydrogel cell cultures was measured using an EdU (5-ethynyl-2'-deoxyuridine) labeling kit (Click-iTTM EdU Cell Proliferation Kit for Imaging, Invitrogen). 5 pM EdU solution was dosed to cells 12 h prior to fixing with 4% paraformaldehyde. Cultures were then permeabilized and stained according to manufacturer’s instructions with 100 pL of stain per well. Plates were then washed and stained with DAPI before proceeding to imaging. EdU staining was done separately from antibodybased staining.
  • references are cited herein throughout using the format of superscripted reference number(s) corresponding to one or more of the following numbered references. For example, citation of references numbers (1) and (2) immediately herein below would be indicated in the disclosure as 1 2 .

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Health & Medical Sciences (AREA)
  • Polymers & Plastics (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Sustainable Development (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Dispersion Chemistry (AREA)
  • Clinical Laboratory Science (AREA)
  • Molecular Biology (AREA)
  • Materials Engineering (AREA)
  • Immunology (AREA)
  • Materials For Medical Uses (AREA)

Abstract

Selon un aspect, la divulgation concerne un appareil comprenant : une plaque ; une pluralité de puits placés dans la plaque, chaque puits individuel de la pluralité de puits s'étendant d'une surface externe de la plaque à une surface interne du puits individuel ; et une pluralité d'hydrogels, au moins deux puits individuels de la pluralité de puits comprenant chacun un hydrogel singulier de la pluralité d'hydrogels ; chaque hydrogel individuel de la pluralité d'hydrogels comprenant un agent de réticulation dithiol et un polymère à premier squelette fonctionnalisé avec un alcène ; et au moins un hydrogel de la pluralité d'hydrogels étant différent des hydrogels restants de la pluralité d'hydrogels. L'invention concerne également des procédés de fabrication de l'appareil. Le présent abrégé est destiné à être utilisé comme outil d'exploration à des fins de recherche dans ce domaine technique particulier, et ne se limite pas à la présente divulgation.
PCT/US2024/050534 2023-10-09 2024-10-09 Appareil de plaque de puits Pending WO2025080682A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202363543189P 2023-10-09 2023-10-09
US63/543,189 2023-10-09

Publications (1)

Publication Number Publication Date
WO2025080682A1 true WO2025080682A1 (fr) 2025-04-17

Family

ID=95396276

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2024/050534 Pending WO2025080682A1 (fr) 2023-10-09 2024-10-09 Appareil de plaque de puits

Country Status (1)

Country Link
WO (1) WO2025080682A1 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20200148804A1 (en) * 2017-05-30 2020-05-14 Mcmaster University Novel Synthetic Polymers and Crosslinked Hydrogel Systems
US20220049294A1 (en) * 2018-12-10 2022-02-17 10X Genomics, Inc. Imaging system hardware
WO2022178181A1 (fr) * 2021-02-17 2022-08-25 University Of Virginia Patent Foundation Influence combinée de repères viscoélastiques et adhésifs sur la prolifération des fibroblastes et la formation d'adhérences focales
US20230135999A1 (en) * 2020-04-03 2023-05-04 The Regents Of The University Of Colorado, A Body Corporate Hybrid-hydrogels comprising decellularized extracellular matrix

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20200148804A1 (en) * 2017-05-30 2020-05-14 Mcmaster University Novel Synthetic Polymers and Crosslinked Hydrogel Systems
US20220049294A1 (en) * 2018-12-10 2022-02-17 10X Genomics, Inc. Imaging system hardware
US20230135999A1 (en) * 2020-04-03 2023-05-04 The Regents Of The University Of Colorado, A Body Corporate Hybrid-hydrogels comprising decellularized extracellular matrix
WO2022178181A1 (fr) * 2021-02-17 2022-08-25 University Of Virginia Patent Foundation Influence combinée de repères viscoélastiques et adhésifs sur la prolifération des fibroblastes et la formation d'adhérences focales

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LEI RUOXING; AKINS ERIN A.; WONG KELLY; REPINA NICOLE; WOLF KAYLA J.; DEMPSEY GARRETT; SCHAFFER DAVID; STAHL ANDREAS; KUMAR SANJAY: "Multiwell combinatorial hydrogel array for high-throughput analysis of cell-ECM interactions", BIOPHYSICAL JOURNAL, ELSEVIER, AMSTERDAM, NL, vol. 121, no. 3, 11 February 2022 (2022-02-11), AMSTERDAM, NL, XP086956467, ISSN: 0006-3495, DOI: 10.1016/j.bpj.2021.11.1416 *

Similar Documents

Publication Publication Date Title
Costa et al. Spheroids formation on non‐adhesive surfaces by liquid overlay technique: Considerations and practical approaches
Hinman et al. In vitro generation of self-renewing human intestinal epithelia over planar and shaped collagen hydrogels
EP2952267B1 (fr) Surfaces superhydrophobes réactives, motifs de telles surfaces et procédés de production et d'utilisation des surfaces superhydrophobes à motifs
ES2952105T3 (es) Matrices de organoides
US20120202263A1 (en) Bioactive Macromers and Hydrogels and Methods for Producing Same
JP6056111B2 (ja) 光分解性架橋剤、光分解性ゲル、細胞培養器具、細胞配列・分別装置、細胞配列方法、細胞分別方法、組織体形成方法および組織体
US20160298087A1 (en) Liver-mimetic device and method for simulation of hepatic function using such device
Serena et al. Soft substrates drive optimal differentiation of human healthy and dystrophic myotubes
KR20160005122A (ko) 개별 세포 배양 미세환경의 어레이, 이러한 어레이의 제조 방법 및 이의 용도
Diederich et al. Bioactive polyacrylamide hydrogels with gradients in mechanical stiffness
Skelton et al. Modular multiwell viscoelastic hydrogel platform for two-and three-dimensional cell culture applications
JP2025179077A (ja) 細胞培養材料
Nava et al. Interactions between structural and chemical biomimetism in synthetic stem cell niches
Varzideh et al. Bioengineering strategies to create 3D cardiac constructs from human induced pluripotent stem cells
Kandasamy et al. Polysulfone membranes coated with polymerized 3, 4-dihydroxy-l-phenylalanine are a versatile and cost-effective synthetic substrate for defined long-term cultures of human pluripotent stem cells
Lei et al. Multiwell combinatorial hydrogel array for high-throughput analysis of cell–ECM Interactions
WO2025080682A1 (fr) Appareil de plaque de puits
WO2022099376A1 (fr) Plate-forme de culture d'organoïdes
Trapani et al. Tunable synthetic hydrogels to study angiogenic sprouting
Angres et al. 3‐D Life biomimetic hydrogels: A modular system for cell environment design
US11767516B2 (en) Methods and compositions for thiol-acrylate based matertals for 3D cell culturing in a microfluidic device
AU2023205537B2 (en) Cell cultivation methodology
WO2020255905A1 (fr) Procédé d'évaluation de substrat de culture et/ou de solution de milieu de culture, et utilisation du procédé d'évaluation
Angres et al. 3-D Life biomimetic hydrogels: A modular system for cell
Dorenvanck Micropatterns on Polyacrylamide Hydrogel for Controlled Cardiomyocyte Attachment using PLL-PEG and ECM Proteins

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24877912

Country of ref document: EP

Kind code of ref document: A1