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WO2024030186A1 - Compositions et procédés associés à des microgels - Google Patents

Compositions et procédés associés à des microgels Download PDF

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WO2024030186A1
WO2024030186A1 PCT/US2023/024353 US2023024353W WO2024030186A1 WO 2024030186 A1 WO2024030186 A1 WO 2024030186A1 US 2023024353 W US2023024353 W US 2023024353W WO 2024030186 A1 WO2024030186 A1 WO 2024030186A1
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peg
kda
microgel
microgels
scaffold
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J. Kent LEACH
Jeremy M. LOWEN
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University of California Berkeley
University of California San Diego UCSD
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University of California San Diego UCSD
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/56—Porous materials, e.g. foams or sponges
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/58—Materials at least partially resorbable by the body

Definitions

  • the present disclosure provides a microgel scaffold comprising: a plurality of polyethylene gly col-vinyl sulfone (PEG-VS); and a plurality of polyethylene glycol-dithiol (PEG-DT) covalently linked to the plurality of PEG-VS, wherein the microgel scaffold has a compressive modulus of between I kPa and 30 kPa. In some embodiments, the microgel scaffold has a porosity of between 20 pm 2 and 100000 pm 2 .
  • the disclosure provides a microgel scaffold comprising: a plurality of polyethylene gly col-vinyl sulfone (PEG-VS); and a plurality of polyethylene glycol-dithiol (PEG-DT) covalently linked to the plurality of PEG-VS, wherein the microgel scaffold has a porosity of between 20 pm 2 and 100000 pm 2 .
  • the microgel scaffold has a compressive modulus of between 1 kPa and 30 kPa.
  • the PEG-VS is selected from the group consisting of 3-arm PEG-VS, 4-arm PEG-VS, 6-arm PEG-VS, and 8-arm PEG-VS.
  • the PEG-VS is an 8-arm PEG-VS.
  • the PEG-VS has a molecular weight ranging from between 2 kDa and 40 kDa.
  • the PEG-DT has a molecular weight ranging from between 0.6 kDa and 35 kDa.
  • the between 40% and 80% are linked to PEG-DT.
  • the microgel scaffold further comprises a plurality of functional moieties conjugated to the PEG-VS.
  • the functional moiety is a peptide (e.g., a cell adhesion peptide).
  • the microgel scaffolds further comprise live cells seeded within the microgel scaffold.
  • the cells comprise human mesenchymal stem cells, induced pluripotent stem cells, embryonic stem cells, osteoblasts, immune cells, blood cells, adipocytes, or combinations thereof.
  • the disclosure provides a method of generating a microgel scaffold, the method comprising: forming a reaction mixture comprising a polyethylene gly col-vinyl sulfone (PEG-VS) and a polyethylene glycol-dithiol (PEG-DT); exposing the reaction mixture to ultraviolet (UV) irradiation for less than 2 minutes (e.g., 1 minute, 1.5 minutes, or 2 minutes) to anneal the PEG-VS and the PEG-DT, thereby forming the microgel scaffold.
  • the PEG-VS is selected from the group consisting of 3-arm PEG-VS, 4-arm PEG-VS, 6-arm PEG-VS, and 8-arm PEG-VS (e.g., 8-arm PEG-VS).
  • the PEG-VS has a molecular weight ranging from between 2 kDa and 40 kDa. In some embodiments, the concentration of the PEG-VS in the reaction mixture is between 1 mM and 80 mM.
  • the PEG-DT has a molecular weight ranging from between 0.6 kDa and 35 kDa. In certain embodiments, the concentration of the PEG-DT in the reaction mixture is between 1 mM and 300 mM. [0015] In some embodiments of the methods described herein, between 40% and 80% (e.g, about 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, or 80%) of the PEG-VS are linked to PEG-DT.
  • 40% and 80% e.g, about 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, or 80%
  • a photoinitiator is added to the reaction mixture prior to the exposing step.
  • the concentration of the photoinitiator in the reaction mixture is between 0.05% w/v and 0.8% w/v.
  • the UV irradiation has a power within the range between 2 mW/cm 2 and 20 mW/cm 2 .
  • the method further comprises, after the exposing step, attaching a functional moiety to the microgel scaffold.
  • the functional moiety is a peptide, e.g., a cell adhesion peptide.
  • the method further comprises, after the exposing step, centrifuging the microgel scaffold at different speeds to achieve a desired porosity of the microgel scaffold.
  • the method further comprises, after the exposing step, seeding the microgel scaffold with live cells.
  • live cells include, but are not limited to, human mesenchymal stem cells, induced pluripotent stem cells, embryonic stem cells, osteoblasts, immune cells, blood cells, adipocytes, or combinations thereof.
  • microgel refers to a three-dimensional hydrogel particle that generally has a diameter between 10 pm - 500 pm .
  • microgel scaffold refers to a network or scaffolding of microgels in which the microgels are crosslinked to each other by way of, for example, covalent bonds.
  • photoinitiator refers to a compound that initiates a polymerization process after irradiation.
  • the photoinitiator can generate acid (a photoacid generator or PAG) or a radical, among other initiating species. The acid, radical, or other species, then initiates a polymerization.
  • biocompatible refers to a material, composition, device, or method that does not have toxic or injurious effects on biological systems. In some medical applications, a biocompatible material, composition, device, or method does not have toxic or injurious effects on a treated subject.
  • FIGS. 1A-1E Microgel annealing process.
  • FIGS. 2A-2G Mechanical properties of microgels and cryopreservation.
  • F Images of cell spreading on frozen and fresh gels compared to non-spread cells on negative control. Scale bar represents 200 pm.
  • FIGS. 3A-3E Modeling void space in between microgels.
  • Statistics Ordinary one-way ANOVA, Tukey’s multiple comparisons test. N >100. *p ⁇ 0.05, **p ⁇ 0.01, and ***p ⁇ 0.001.
  • FIGS. 4A-4D Microgels as an aggregate-forming platform.
  • FIGS. 5A-5D Spheroids spread rapidly in large and small microgel scaffolds.
  • A) DAPI/Phalloidin stains illustrate that void space inherent in microgel scaffolds facilitates rapid spreading in large and small microgel scaffolds. Inset scale bars represent 200 pm. Primary picture scale bars represent 1 mm.
  • D) alamarBlue assay confirms that both scaffolds promote high cell metabolic activity.
  • FIGS. 6A-6G Microgel size influences macrophage polarization.
  • D,G M2 polarization is greater in large microgel scaffolds. Scale bars represent 100 pm.
  • FIGS. 7A-7C Subfascial implantation of microgel scaffolds.
  • FIG. 8 Storage modulus of microgel scaffolds increases over time when exposed to UV light.
  • the extracellular matrix of cells plays a vital role in processes such as differentiation, angiogenesis, proliferation, invasion, and wound repair. To successfully undergo these processes cells must be able to remodel their surrounding environment to create space for migration and protein deposition. Described herein are highly tunable microgels with size, stiffness, and biochemical presentation modified to influence cell fate. Ultraviolet light is utilized to anneal the microgels together, creating a scaffold with microporous void space throughout. By altering microgel diameter, it is possible to control void space size and therefore the amount of cell spreading and aggregation throughout the annealed microgel scaffold. This void space allows for rapid infiltration without cells having to remodel the surrounding environment or wait for a porogen to dissolve.
  • microgels to be noninvasively injected into a desired mold or wound defect before annealing, and microgels of different properties can combined to create a heterogenous scaffold. This approach is clinically relevant given its tunability to many tissue types and fast annealing time, e.g., under 1 minute.
  • Tunable and noninvasive scaffolds to direct cell fate are desired in many tissue engineering applications such as wound healing, organoid systems, and drug delivery.
  • tissue engineering applications such as wound healing, organoid systems, and drug delivery.
  • Microgels are an emerging tool which fulfill this roll given their modularity, injectability, and range of fabrication techniques.
  • a plethora of studies have harnessed microgels to study cell behavior in response to stiffness, degradability, and biochemical cues.
  • a range of materials are commonly used to synthesize microparticles including alginate, poly(ethylene) glycol (PEG), and hyaluronic acid. Given the vast amount of materials and tunability it is possible to synthesize microgels to meet a variety of scaffold specifications.
  • polymeric scaffolds are composed of bulk hydrogels that exhibit a nanoporous mesh size. This pore size prevents infiltration by cells and hinders biological activity.
  • MMP matrix metalloproteinase
  • An advantage to microparticle-based scaffold is the void space which inherently exists between the particles. This permits cells to immediately migrate without having to remodel their surrounding environment. Previous work has illustrated that human dermal fibroblasts proliferated over twice as much in a microgel scaffold compared to a bulk hydrogel scaffold.
  • microgels overcome this challenge by decoupling scaffold formation from hydrogel crosslinking. It is also necessary' to create an incision to insert bulk hydrogels a defect site subcutaneously.
  • Microgel scaffolds overcome this limitation with injectable microporous scaffolds out-performing their non-porous controls in wound healing. Microgels are also able to maintain their spatial orientation when loaded and extruded from a syringe, and have the advantage of naturally filling up the dimensions of the defect site.
  • microgels While many microgel platforms rely on chemical assembly methods such as enzymatic catalysis or click chemistry , demonstrated herein is an ultraviolet (UV) method of annealing microgels. This method is effective for microgels of various sizes, e.g., ranging from about 45 pm to about 140 pm in diameter and requires as little as one minute for annealing. Microgels spanning from about 10 kPa to 80 kPa were generated. Their compressive modulus was compared to to bulk gels using a MicroTester. Further, the compressive modulus and bioactivity of the microgels remained constant after being cryopreserved at -20 °C.
  • UV ultraviolet
  • a custom MATLAB code was also developed to predict void space diameter based on theoretical and observed microgel diameters.
  • the ability to alter porosity was utilized to influence macrophage polarization.
  • This platform allows ability to predictability alter cell spreading and aggregation was also demonstrated.
  • the ease of recovering cells from this system was also utilized to run flow cytometry to examine polarization state of the macrophages.
  • the conditioned media from the macrophage seeded scaffolds was then collected to influence mesenchymal stem cell (MSC) differentiation.
  • MSC mesenchymal stem cell
  • the present disclosure provides a microgel scaffold that is made from a plurality of polyethylene gly col-vinyl sulfone (PEG-VS) and a plurality of polyethylene glycol-dithiol (PEG-DT) covalently linked to the plurality of PEG-VS.
  • the microgel scaffold described herein has a compressive modulus of between 1 kPa and 30 kPa and/or a porosity of between 20 pm 2 and 100000 pm 2 .
  • the disclosure also provides methods of making the microgel scaffold that utilizes ultraviolet (UV) irradiation.
  • UV ultraviolet
  • the microgel scaffold of the present disclosure comprises a plurality of polyethylene gly col -vinyl sulfone (PEG-VS); and a plurality of polyethylene glycol-dithiol (PEG-DT) covalently linked to the plurality of PEG-VS.
  • PEG-VS polyethylene gly col -vinyl sulfone
  • PEG-DT polyethylene glycol-dithiol
  • the PEG-VS used can contain different numbers of arms depending on the desired degree of crosslinking between PEG-VS and PEG-DT.
  • the PEG-VS is selected from the group consisting of 3-arm PEG-VS, 4-arm PEG-VS, 6-arm PEG-VS, and 8-arm PEG-VS.
  • the PEG-VS is an 8-arm PEG-VS.
  • the PEG- VS has a molecular weight ranging from between 2 kDa and 40 kDa (e.g, between 2 kDa and 35 kDa, between 2 kDa and 30 kDa, between 2 kDa and 25 kDa, between 2 kDa and 20 kDa, between 2 kDa and 15 kDa, between 2 kDa and 10 kDa, between 2 kDa and 5 kDa, between 5 kDa and 40 kDa, between 10 kDa and 40 kDa, between 15 kDa and 40 kDa, between 20 kDa and 40 kDa, between 25 kDa and 40 kDa, between 30 kDa and 40 kDa, or between 35 kDa and 40 kDa; 2 kDa, 4 kDa, 6 kDa, 8 kDa, 10 kDa, 12 kDa; 2
  • the PEG-DT has a molecular weight ranging from between 0.6 kDa and 35 kDa (e.g, between 0.6 kDa and 30 kDa, between 0.6 kDa and 25 kDa, between 0.6 kDa and 20 kDa, between 0.6 kDa and 15 kDa, between 0.6 kDa and 10 kDa, between 0.6 kDa and 5 kDa, between 0.6 kDa and 3 kDa, between 0.6 kDa and 1 kDa, between 1 kDa and 35 kDa, between 5 kDa and 35 kDa, between 10 kDa and 35 kDa, between 15 kDa and 35 kDa, between 20 kDa and 35 kDa, between 25 kDa and 35 kDa, or between 30 kDa and 35 kDa; 0.6 kDa, 1
  • the microgel scaffolds described herein are highly tunable and can be constructed with different sizes of porosity within the microgel scaffolds.
  • the porosity of the microgel scaffolds can be tuned depending on the use purpose and allows rapid cell infiltration into the microgel scaffolds without the cells needing to remodel their surrounding environment.
  • the microgel scaffold comprises a porosity of between 20 pm 2 and 100000 pm 2 (e.g., between 20 pm 2 and 90000 pm 2 , between 20 pm 2 and 80000 pm 2 , between 20 pm 2 and 70000 pm 2 , between 20 pm 2 and 60000 pm 2 , between 20 pm 2 and 50000 pm 2 , between 20 pm 2 and 40000 pm 2 , between 20 pm 2 and 30000 pm 2 , between 20 pm 2 and 20000 pm 2 , between 20 pm 2 and 10000 pm 2 , between 20 pm 2 and 9000 pm 2 , between 20 pm 2 and 8000 pm 2 , between 20 pm 2 and 7000 pm 2 , between 20 pm 2 and 6000 pm 2 , between 20 pm 2 and 5000 pm 2 , between 20 pm 2 and 4000 pm 2 , between 20 pm 2 and 3000 pm 2 , between 20 pm 2 and 2000 pm 2 , between 20 pm 2 and 1000 pm 2 , between 20 pm 2 and 900 pm 2 , between 20 pm 2 and 800 pm 2 , between 20 pm 2 and 700 , between 20 pm
  • the microgel scaffold comprises a porosity of between 30 pm 2 and 100000 pm 2 (e.g., between 40 pm 2 and 100000 pm 2 , between 50 pm 2 and 100000 pm 2 , between 60 pm 2 and 100000 pm 2 , between 70 pm 2 and 100000 pm 2 , between 80 pm 2 and 100000 pm 2 , between 90 pm 2 and 100000 pm 2 , between 100 pm 2 and 100000 pm 2 , between 200 pm 2 and 100000 pm 2 , between 300 pm 2 and 100000 pm 2 , between 400 pm 2 and 100000 pm 2 , between 500 pm 2 and 100000 pm 2 , between 600 pm 2 and 100000 pm 2 , between 700 pm 2 and 100000 pm 2 , between 800 pm 2 and 100000 pm 2 , between 900 pm 2 and 100000 pm 2 , between 1000 pm 2 and 100000 pm 2 , between 2000 pm 2 and 100000 pm 2 , between 3000 pm 2 and 100000 pm 2 , between 4000 pm 2 and 100000 pm 2 , between 5000 pm 2 and 100000 pm 2 (e.g.,
  • the microgel scaffold described herein can have a compressive modulus of between 1 kPa and 30 kPa (e.g., between 1 kPa and 28 kPa, between 1 kPa and 26 kPa, between 1 kPa and 24 kPa, between 1 kPa and 22 kPa, between 1 kPa and 20 kPa, between 1 kPa and 18 kPa, between 1 kPa and 16 kPa, between 1 kPa and 14 kPa, between 1 kPa and 12 kPa, between 1 kPa and 10 kPa, between 1 kPa and 8 kPa, between 1 kPa and 6 kPa, between 1 kPa and 4 kPa, between 1 kPa and 2 kPa, between 2 kPa and 30 kPa, between 4 kPa and 30 kPa, between 6 kPa and 30 kPa
  • the PEG-VS in the microgel scaffold described herein can be modified with different functional moieties.
  • the functional moieties are used to direct and influence cell seeding, attachment, spreading, and/or growth as the cells are injected into the microgel scaffold.
  • the functional moieties can also direct and influence cell fate and differentiation.
  • Examples of functional moieties that can be attached to the PEG-VS include, but are not limited to, a peptide, an oligonucleotide, a fluorescent label, and/or a carbohydrate.
  • the PEG-VS in the microgel scaffold can be modified with a peptide.
  • the peptide can be a cell adhesion peptide, which promotes cell adhesion and is often derived from extracellular matrix glycoproteins such as laminin, fibronectin, and collagen.
  • cell adhesion peptides can be attached to PEG-VS include, but are not limited to, RGDSPGERCG (SEQ ID NO: 1), HAVDIGGGC (SEQ ID NO:2), LNIVSVNGRH (SEQ ID NO:3), DNRIRLQAK (SEQ ID NO:4), KATPMLKMRTSFHGCIK (SEQ ID NO:5), KEGYKVRLDLNITLEFRTTSK (SEQ ID NO:6), KNLEISRSTFDLLRNSYGVRK (SEQ ID NO:7), KQNCLSSRASFRGCVRNLRLSR (SEQ ID NO:8), KQKCLRSQTSFRGCLRKLALIK (SEQ ID NOV), CRNRGRCNSSLFQVRSRKLLSA (SEQ ID NO: 10),
  • the cell adhesion peptide has the sequence of RGDSPGERCG (SEQ ID NO: 1). In particular embodiments, the cell adhesion peptide has the sequence of HAVDIGGGC (SEQ ID NO:2). Additional cell adhesion peptides are described in, e.g, Huettner et al., Trends Biotechnol 2018 Apr;36(4):372-383; Ruoslahti Annu Rev Cell Dev Biol 1996;12:697-715; and LeBaron and Athanasiou Tissue Eng 2000 Apr;6(2):85-103.
  • the microgel scaffold of the present disclosure can be seeded with different types of cells.
  • the cells can be, for example and without limitation, human mesenchymal stem cells (hMSCs), induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), osteoblasts and osteoblast precursors (e..g, MC3T3 cells), immune cells (e.g., macrophages), blood cells, adipocytes, and others.
  • the cells seeded in the microgel scaffold are live cells.
  • the seeding of the microgel scaffold with cells can occur prior to the injecting of the microgel scaffold into its destination, e.g., tissue and/or bone defects.
  • the seeding of the microgel scaffold with cells can occur subsequent to the injecting of the microgel scaffold into its destination, e.g., tissue and/or bone defects.
  • a reaction mixture comprising a plurality of polyethylene glycol -vinyl sulfone (PEG-VS) and a plurality of polyethylene glycol-dithiol (PEG-DT) is formed.
  • PEG-VS polyethylene glycol -vinyl sulfone
  • PEG-DT polyethylene glycol-dithiol
  • the UV irradiation of the reaction mixture to form the microgel scaffold is about 30 seconds, 40 seconds, or 50 seconds. In some embodiments, the UV irradiation of the reaction mixture to form the microgel scaffold is about 60 seconds (1 minute), 70 seconds, 80 seconds, 90 seconds, 100 seconds, 110 seconds, or 120 seconds (2 minutes).
  • the power of the UV irradiation is between 2 and 20 mW/cm 2 , e.g., 2 mW/cm 2 , 4 mW/cm 2 , 6 mW/cm 2 , 8 mW/cm 2 , 10 mW/cm 2 , 12 mW/cm 2 , 14 mW/cm 2 , 16 mW/cm 2 , 18 mW/cm 2 , or 20 mW/cm 2
  • the methods of generating the microgel scaffolds described herein use 1 minute UV irradiation at 20 mW/cm 2 .
  • the methods of generating the microgel scaffolds described herein use 1.5 minute UV irradiation at 20 mW/cm 2 . In particular embodiments, the methods of generating the microgel scaffolds described herein use 2 minutes UV irradiation at 20 mW/cm 2 .
  • the concentration of the PEG-V S in the reaction mixture is between 1 mM and 80 mM (e.g., between 1 mM and 70 mM, between 1 mM and 60 mM, between 1 mM and 50 mM, between 1 mM and 40 mM, between 1 mM and 30 mM, between 1 mM and 20 mM, between 1 mM and 10 mM, between 1 mM and 5 mM, between 5 mM and 80 mM, between 10 mM and 80 mM, between 20 mM and 80 mM, between 30 mM and 80 mM, between 40 mM and 80 mM, between 50 mM and 80 mM, between 60 mM and 80 mM, or between 70 mM and 80 mM; 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM;
  • the concentration of the PEG-DT in the reaction mixture is between 1 mM and 300 mM (e.g., between 1 mM and 280 mM, between 1 mM and 260 mM, between 1 mM and 240 mM, between 1 mM and 220 mM, between 1 mM and 200 rnM, between 1 mM and 180 mM, between 1 mM and 160 mM, between 1 mM and 140 mM, between 1 mM and 120 mM, between 1 mM and 100 mM, between 1 mM and 80 mM, between 1 mM and 60 mM, between 1 mM and 40 mM, between 1 mM and 20 mM, between 1 mM and 10 mM, between 10 mM and 300 mM, between 20 mM and 300 mM, between 40 mM and 300 mM, between 60 mM and 300 mM, between 80 mM and 300 mM,
  • the concentration of the PEG-DT in the reaction mixture is about 1 mM, 10 mM, 20 mM, 40 mM, 60 mM, 80 mM, 100 mM, 120 mM, 140 mM, 160 mM, 180 mM, 200 mM, 220 mM, 240 mM, 260 mM, 280 mM, or 300 mM).
  • the size of the porosity, or void space, within the microgel scaffolds can be changed easily by incorporating PEG-V S of different arms, as well as altering the amount of PEG-DT and the ratio of PEG-V S to PEG-DT in the reaction mixture to tune the degree of crosslinking of PEG-VS and PEG-DT.
  • the microgel scaffold after the exposing the reaction mixture containing PEG-VS and PEG-DT to UV irradiation, can be centrifuged at different speeds to achieve a desired porosity of the microgel scaffold.
  • a higher centripetal force e.g, a centripetal force of at least 15,000 g
  • a centripetal force can be used to reduce the porosity of the microgel scaffold.
  • sacrificial materials can be added to increase the porosity of the microgel scaffold.
  • sacrificial materials include, but are not limited to, salts, sugars, paraffin, and gelatin.
  • sacrificial materials can be removed by immersing the scaffold in a solvent which dissolves the porogen, or increasing the temperature to dissolve the porogen.
  • a photoinitiator is added to the reaction mixture prior to exposing the reaction mixture to UV irradiation.
  • the concentration of photoinitiator in the reaction mixture to generate the microgel scaffolds can be within the range between 0.05% w/v and 0.8% w/v.
  • the concentration of photoinitiator can be within the range between 0.05% w/v and 0.6% w/v, between 0.05% w/v and 0.5% w/v, between 0.05% w/v and 0.4% w/v, between 0.05% w/v and 0.3% w/v, between 0.05% w/v and 0.2% w/v, or between 0.05% w/v and 0.1% w/v.
  • the concentration of photoinitiator can be within the range between 0.1% w/v and 0.8% w/v, between 0.2% w/v and 0.8% w/v, between 0.3% w/v and 0.8% w/v, between 0.4% w/v and 0.8% w/v, between 0.5% w/v and 0.8% w/v, between 0.6% w/v and 0.8% w/v, or between 0.7% w/v and 0.8% w/v.
  • Different photoinitiators are sensitive to different wavelenghts, such as wavelengths in the UV spectrum (e.g, from about 100 to about 400 nm) and wavelengths in the visible light spectrum (e.g., from about 400 to about 800 nm).
  • photoinitiators include, but are not limited to, eosin Y, VA-086, riboflavin, carboxylated camphorquinone, rose bengal, erythrosine, WSPI, BDEA, 2PCK, G2CK, Irgacure 2959, Irgacure 184, Irgacure 651, Irgacure 369, Irgacure 907, PEGDA, MeHA, GelMA, MAPO (TPO), TPO-Na, LAP, BAPO, BAPO-Ona, and BAPO-OLi. Photoinitiators are also described in, e.g., Tomal and Ortyl, Polymers (Basel).
  • the methods of generating microgel scaffolds described herein use a plurality of PEG-VS selected from the group consisting of 3-arm PEG-VS, 4- arm PEG-VS, 6-arm PEG-VS, and 8-arm PEG-VS.
  • the PEG-VS is an 8-arm PEG-VS.
  • the PEG-VS used in the methods can have a molecule weight ranging from between 2 kDa and 40 kDa (e.g., between 2 kDa and 35 kDa, between 2 kDa and 30 kDa, between 2 kDa and 25 kDa, between 2 kDa and 20 kDa, between 2 kDa and 15 kDa, between 2 kDa and 10 kDa, between 2 kDa and 5 kDa, between 5 kDa and 40 kDa, between 10 kDa and 40 kDa, between 15 kDa and 40 kDa, between 20 kDa and 40 kDa, between 25 kDa and 40 kDa, between 30 kDa and 40 kDa, or between 35 kDa and 40 kDa; 2 kDa, 4 kDa, 6 kDa, 8 kDa, 10 kDa, 12 kD
  • the methods of generating microgel scaffolds described herein use a plurality of PEG-DT, in which the PEG-DT has a molecular weight ranging from between 0.6 kDa and 35 kDa (e.g., between 0.6 kDa and 30 kDa, between 0.6 kDa and 25 kDa, between 0.6 kDa and 20 kDa, between 0.6 kDa and 15 kDa, between 0.6 kDa and 10 kDa, between 0.6 kDa and 5 kDa, between 0.6 kDa and 3 kDa, between 0.6 kDa and 1 kDa, between 1 kDa and 35 kDa, between 5 kDa and 35 kDa, between 10 kDa and 35 kDa, between 15 kDa and 35 kDa, between 20 kDa and 35 kDa, between 25 kDa and 35 kDa, between a mole
  • methods of generating a microgel scaffold comprising: forming a reaction mixture comprising a plurality of an 8-arm PEG-V S and a plurality of a PEG-DT; exposing the reaction mixture to UV irradiation for less than 2 minutes (e.g., 1 minute, 1.5 minutes, or 2 minutes) to anneal the PEG-V S and the PEG-DT, thereby forming the microgel scaffold.
  • the PEG-DT used in the methods is about 3.5 kDa.
  • the PEG-DT used in the methods is about 4-5 mM (e.g., 5 mM).
  • an eosin Y photoinitiator is added to the reaction mixture prior to exposing the reaction mixture to UV irradiation.
  • a VA-086 photoinitiator is added to the reaction mixture prior to exposing the reaction mixture to UV irradiation.
  • the microgel scaffolds described herein are readily amenable to use with different of cell types and for a variety of clinical relevant applications.
  • the high tunability of the microgel scaffolds described herein makes the microgel scaffolds easily tailored to a diverse range of cell types of different sizes and attachment, migration, and growth patterns.
  • the size of the porosity, or void space, within the microgel scaffolds can be changed easily by incorporating PEG-VS of different arms, as well as altering the amount of PEG-DT and the ratio of PEG-VS to PEG-DT in the reaction mixture to tune the degree of crosslinking of PEG-VS and PEG-DT.
  • cells can be seeded in microgel scaffolds described herein are mammalian cells, although the cells may be from different species (e.g., humans, mice, rats, primates, pigs, and the like).
  • the cells can be primary cells, or they may be derived from an established cellline.
  • Cells can be from multiple donor types, can be progenitor cells, tumor cells, and the like.
  • the cells are freshly isolated cells (for example, seeded within 24 hours of isolation), e g. , freshly isolated cells from donor organs
  • the cells are isolated from individual donor organs and an assembled tissue construct is specific for that donor.
  • cell types which may be seeded in the microgel scaffolds described herein include pancreatic cells (alpha, beta, gamma, delta), enterocytes, renal epithelial cells, astrocytes, muscle cells, brain cells, neurons, glia cells, respiratory epithelial cells, lymphocytes, erythrocytes, blood-brain barrier cells, kidney cells, cancer cells, normal or transformed fibroblasts, liver progenitor cells, oval cells, adipocytes, osteoblasts, osteoclasts, myoblasts, beta-pancreatic islets cells, stem cells (e.g., embryonic stem cells, hematopoietic stem cells, mesenchymal stem cells, endothelial stem cells, etc.), myocytes, keratinocytes, and indeed any cell type that adheres to a substrate.
  • pancreatic cells alpha, beta, gamma, delta
  • enterocytes enterocytes
  • renal epithelial cells enterocytes
  • astrocytes astrocytes
  • the microgel scaffolds described herein can include peptides or proteins from the extracellular matrix (ECM), i.e., an ECM peptide or protein conjugated to the PEG-VS.
  • ECM peptides and proteins found in the cell’s native microenvironment are useful in maintaining attachment, growth, migration, and/or function of the cell.
  • An ECM peptide or protein can be a cell adhesion peptide or protein that is specific to the cell type to be seeded in the microgel scaffolds.
  • cell adhesion peptides can be attached to PEG-VS include, but are not limited to, RGDSPGERCG (SEQ ID NO:1), HAVDIGGGC (SEQ ID NO:2), LNIVSVNGRH (SEQ ID NO:3), DNRIRLQAK (SEQ ID NO:4), KATPMLKMRTSFHGCIK (SEQ ID NO:5), KEGYKVRLDLNITLEFRTTSK (SEQ ID NO: 6), KNLEISRSTFDLLRNSYGVRK (SEQ ID NO: 7),
  • the cell adhesion peptide has the sequence of RGDSPGERCG (SEQ ID NO: 1).
  • the cell adhesion peptide has the sequence of HAVDIGGGC (SEQ ID NO:2).
  • ECM peptides and proteins include, but are not limited to, collagen I, collagen III, collagen IV, laminin, and fibronectin.
  • the cells that can be seeded into the microgel scaffolds described herein include, but are not limited to, human mesenchymal stem cells, induced pluripotent stem cells, embryonic stem cells, osteoblasts, immune cells (e.g., macrophages), blood cells (e.g., redblood cells), adipocytes, or combinations thereof.
  • the microgel scaffolds described herein can be injected into the desired mold or wound defect before annealing using UV irradiation. In other embodiments, the microgel scaffolds described herein can be injected into the desired mold or wound defect after annealing using UV irradiation. Particularly, in some embodiments, the cells can be seeded into the microgel scaffolds after the microgel scaffolds are injected into the desired mold or would defect. In other embodiments, the cells can be seeded into the microgel scaffolds before the microgel scaffolds are injected into the desired mold or would defect.
  • microgel scaffolds of different porosity and/or with different cell types seeded can be combined to construct a heterogeneous microgel scaffolds.
  • Microgels were synthesized using a previously established high-throughput microfluidic device. To facilitate microgel annealing we produced microgels with only a fraction of the arms crosslinked to enable the remaining free arms to be used for photoannealing (FIG. 1A). We stoichiometrically controlled the number of free arms by altering the ratio of 8-arm PEG-vinyl sulfone (PEG-VS) to the crosslinker PEG-dithiol (PEG- DT). By using the vinyl sulfone moiety for both microgel crosslinking and annealing we avoid the use of additional complex or expensive reagents used in methods such as enzymatic catalysis or host-guest interactions. The use of an 8-arm PEG allows us to fine tune the number of arms used for crosslinking while leaving arms available for biofunctionalization such as the addition of peptides.
  • PEG-VS 8-arm PEG-vinyl sulfone
  • PEG-DT crosslinker PEG
  • microgels To anneal the microgels we added additional PEG-DT in a solution of HEPES with photoinitiator (FIG. IB). The microgels were then spun down and supernatant removed. For larger microgels a higher centripetal force is required to effectively jam the microgels due to the increased voice space in between them.
  • the aggregated microgels can be mixed with cells or other additives as the microgels exhibit shear thinning behavior and can easily be manipulated.
  • the microgel slurry was plated and exposed to UV light to finish the annealing process. To determine necessary time for annealing, we measured the storage modulus of microgel slurries during UV exposure (FIG. 8).
  • microgel scaffolds that were of clinically relevant thickness (FIG. IE).
  • FOG. IE clinically relevant thickness
  • the 1 cm thick microgel scaffold was able to withstand cyclically applied compression, demonstrating the strength of the annealing.
  • This method of annealing microgels was aimed to minimize the steps needed for scaffold formation. By creating microgels which only require a UV light source to crosslink the goal is to increase the accessibility of this platform. By stoichiometrically controlling the number of arms used for crosslinking and annealing we minimize the number of reagents needed to form an annealed microgel scaffold.
  • microgels The ability of the microgels to be cryopreserved is important for long-term storage and clinical application.
  • microgels By measuring the mechanical properties of the microgels and demonstrating their ability to be cryopreserved we further illustrated how they can be useful in the clinic.
  • the large range of stiffnesses along with peptide bioactivity permit these microgels to be tailored for a variety of tissue types.
  • the microgels demonstrated their ability to maintain a constant compressive modulus and peptide function after cryopreservation, opening the door for on- demand use in the clinic.
  • Modeling the porosity of our microgel scaffolds is crucial for predicting how cells may migrate, aggregate, and differentiate within our constructs. Smaller microgels will have less void space in between them and therefore cells will be increasingly monodispersed with less room to spread out. Conversely, larger microgels will have increased void space in between them so cells will be able to more easily migrate and aggregate. Similarly, the more poly disperse microgels are the larger the void space will be due to the microgels being unable to optimally pack together. To model these phenomena we utilized custom MATLAB code along with FIJI to predict how actual or theoretical batches of microgels may pack together.
  • the ability to model void space is beneficial for many cell-based projects which can vary greatly in the scale of biologies used. There are significant differences between cell types such as red blood cells and adipocytes which can range from 7.5 pm to over 100 pm in diameter, respectively. Aggregates of cells such as mesenchymal spheroids can reach up to 600 pm without a hypoxic core. It is also useful for the incorporation of other additives such as drug-loaded nanoparticles. Overall, modeling is a powerful tool that can be utilized to fabricate microgels for a specific porosity to influence cell phenotype.
  • Multicellular spheroids can act as building blocks that capture complex aspects of in vivo environments and represent improved model systems to study development and disease. Spheroids exhibit increased viability along with enhanced proangiogenic, anti-inflammatory, and tissue-forming potential. Thus, the development of a platform that can promote the formation of cellular aggregates would be useful for many tissue engineering applications.
  • To further investigate the impact of microgel size on cellular aggregate formation we seeded monodisperse MSCs in small and large microgel scaffolds lacking adhesive moieties to encourage cell-cell adhesion and promote aggregate formation. We observed spheroid formation over the first 48 hours, which is a typical timeframe for spheroid formation using other methods such as hanging drop or formation in non-adhesive well plates.
  • a scaffold which promotes spheroid formation may eliminate the need to form spheroids a priori, which commonly requires a minimum of 48 hours. Given the correlation between our model of void space and aggregate size, it would be possible to design specific size microgels for formation of a desired spheroid size.
  • microgels allow for rapid cell infiltration and proliferation without cells needing to remodel the surrounding environment, as is required in bulk hydrogels.
  • spheroids in scaffolds composed of large and small microgels. By seeding the microgels with spheroids, we can examine migration distance and density which is not possible if the cells were monodisperse.
  • Microgels increase MSC retention and proliferation compared to traditional nanoporous hydrogels. However, the influence of altering microgel diameter on MSC growth has not been investigated. We hypothesized the increased surface area present in small microgel scaffolds would promote faster cell migration from the spheroid into the scaffold.
  • both large and small microgels promoted rapid migration of cells into the scaffold.
  • the leading edge of cell migration was comparable for both microgel sizes on Days 1 and 7 (FIG. 5B).
  • the greater porosity within large microgel scaffolds did not significantly hinder cell migration.
  • the smaller diameter microgel scaffold had a higher cell density on Days 1 and 7 (FIG. 5C). This can be attributed to the higher surface area-to-volume ratio of the smaller microgels that provide more attachment sites for cell spreading.
  • Both scaffolds promoted similar levels of metabolic activity when normalized to DNA, indicating both formulations promote high viability (FIG. 5D).
  • the rapid migration of cells in both conditions highlights the advantage of inherent porosity in microgel scaffolds.
  • the measurable migration on Day 1 reflects the cells ability to immediately migrate without first remodeling the surrounding environment. While void space size is significantly smaller in small microgel scaffolds compared to large microgel scaffolds, it was sufficient to permit cell movement. Future work could utilize different size microgels to regulate the density of cell infiltration and spreading.
  • Solid biomaterial implants often induce a foreign body response, regulated by macrophages, that is characterized by poor vascularization and fibrosis.
  • a biomaterial will promote a pro-regenerative response characterized by cell infiltration and material integration.
  • Microgels can promote a pro-regenerative M2 phenotype compared to clinical controls such as Oasis Wound Matrix decellularized ECM.
  • Oasis Wound Matrix decellularized ECM Oasis Wound Matrix decellularized ECM.
  • Macrophages were collected after 6 days in culture for assessment of polarization via flow cytometry.
  • a subset of macrophages was stained with CellTrace to permit visualization with fluorescent microscopy and observe their interaction with the microgel scaffolds.
  • Microgels are a promising candidate for porous biomaterials given the tunable void space that exists between them. While the porosity in the small microgel scaffold was large enough to promote rapid and dense spreading as demonstrated previously, it may be so small as to promote unintended effects such as a pro-inflammatory response from macrophages. Therefore, when picking a microgel size, it is important to consider the resultant porosity between them.
  • Hematoxylin and eosin (H&E) staining revealed that while cells infiltrated both scaffolds, they tended to migrate farther inside the larger microgel scaffolds (FIG. 7C). Where cells did infiltrate, they surround both sizes of microgels which indicates the porosity in both scaffolds was sufficient for migration. The reduced migration depth in the smaller microgel scaffolds could be a result of the smaller porosity hindering migration or the increased surface area resulting in cells spreading out more densely but not as far. Masson’s trichrome staining corroborated the increased migration seen in the large microgel scaffolds (FIG. 7C). Larger aggregates of cells are present between the larger microgels.
  • collagen is present primarily around the surface of the implants but is scarce in between the microgels themselves. While this indicates a FBR to the PDMS mold in which the microgel scaffolds were housed, it appears the FBR to the microgels themselves was minimized.
  • microgels In vivo implantation resulted in robust endogenous cell spreading and infiltration in our microgel scaffolds.
  • the increased surface area of the smaller microgels resulted in denser spreading near the surface of the scaffold, but less migration into the scaffold.
  • cellular aggregates in the larger microgel scaffold consistently migrated the depth of the scaffold but were more spread out.
  • Device Fabrication Soft lithography was used to create microfluidic master molds on silicon wafers (University Wafer) for devices first described in Rutte et al. We used a two-layer photolithography process with SU-8 10 and SU-8 100 (Kayaku Advanced Materials) to create channels heights for our different sized microgels. The layers were aligned utilizing a EVG 620-mask aligner. A Bruker Dektak XT was used to verify the heights of our device channels. A nozzle channel height of 12 pm and 38 pm created droplets of ⁇ 48 pm and 146 pm, respectively.
  • PDMS Poly dimethylsiloxane
  • Sylgard 184 Poly dimethylsiloxane
  • the PDMS mixture was desiccated and cured at 65°C for at least an hour and cut out.
  • PDMS devices and glass slides were plasma cleaned and bonded together followed by a bake of 125°C on a hotplate for 1 hour.
  • the microgel devices were then treated with Aquapel and Novec 7500 Oil (3M) to render the devices hydrophobic and fluorophilic, respectively.
  • Microgel Fabrication Our microgel devices contained a continuous oil phase and a dispersed aqueous phase.
  • the oil phase consisted of Novec 7500 Oil and .75% wt Picosurf (Sphere Fluidics) for the large microgels. Picosurf concentration was increased to 2% wt for the small microgels.
  • the aqueous phase consisted of 8-arm PEG-VS (JenKem) in 0.15 M triethanolamine (TEO A, pH 5.1, Sigma) buffer and 3.5 kDa PEG-DT (JenKem).
  • the solutions were injected into the microfluidic using syringe pumps (NE-1000 and World Precision Instruments) with the continuous phase set at twdce the flow rate of the dispersed phase.
  • microgels After exiting the device microgels were combined with a solution of 1% v/v tnethylamine (TEA, Sigma)) in Novec 7500 Oil using a Y-junction (IDEX Health and Science). The microgels were left at room temperature overnight to ensure complete crosslinking.
  • Microgel Cleaning To clean the microgels first excess oil was removed by pipetting. A solution of 20 wt% 1H,1H,2H,2H-Perfluoro-1 -octanol (Sigma) in Novec 7500 Oil approximately equal to the volume of remaining microgels was then added to break the emulsion. HEPES buffer (25 mM, pH 7.4) was then added to swell and disperse the microgels. A hexane wash w as then repeated 3x to remove the remaining oil. Enough hexane was added to pellet the microgels at the bottom after being spun down at 2000g. For cell experiments the microgels were then washed 3x with 70% ethanol for sterilization.
  • Microgel Annealing To anneal the microgels they were first spun down at 2000g and excess liquid removed. The microgels were then resuspended in a solution of 5mM PEG- DT in HEPES containing 0.4% VA-086 photoinitiator (FUJIFILM) equal to the volume of microgels. After incubating for at least 1 minute, the microgels were spun down for 3 minutes at 3000g for the small microgels and 15,000g for the larger microgels. The supernatant was then removed and microgels plated in the desired mold utilizing a positive displacement pipette (Gilson). The microgel slurry was then exposed to UV light (20 mW/cm2, Omnicure S2000) for 2 minutes to form annealed scaffolds.
  • UV light 20 mW/cm2, Omnicure S2000
  • Microgels were created such that 60,70, and 80% of the PEG-VS arms were crosslinked with PEG-DT. Microgels were then soaked in a solution containing 0.4% photoinitiator in HEPES, HEPES + PEG-DT, HEPES + NVP (Sigma), or HEPES + PEG-DT + NVP. Microgel scaffolds were then formed as detailed previously and mechanically testing was performed and described below.
  • the compressive modulus was calculated over the course of compression using formulas descnbed in Kim et al.
  • the linear region of the of compressive modulus vs nominal strain graph was recorded as the calculated modulus.
  • Storage modulus of microgel scaffolds over time was measured using a Discovery HR2 Rheometer (TA Instruments, New Castle, DE) with a stainless steel, cross hatched, 8 mm plate geometry.
  • the scaffolds were exposed to U V light after 30 s, while it remained off the entire time for the control group.
  • a custom oscillatory time sweep 1% strain, 1 rad s 1 angular frequency
  • ECFCs Human endothelial colony forming cells
  • UMBCP UC Davis Cord Blood Collection Program
  • ECFCs were expanded in EGM-2 supplemented media (PromoCell, Heidelberg, Germany) with gentamicin (50 pg mL’ 1 ; ThermoFisher, Waltham, MA) and amphotericin B (50 ng mL' 1 ; ThermoFisher) under standard culture conditions (37°C, 5% CO2, 21% O2) until use at passages 7-8. Media changes were performed every 2 days.
  • GM growth medium
  • a-MEM minimum essential alpha medium
  • FBS fetal bovine serum
  • penicillin/streptomycin Gamini Bio-Products, Sacramento, CA
  • the cell actin cytoskeleton was stained with Alexa Fluor 488 Phalloidin solution (Thermo Fisher; 1:400 in PBS), and cell nuclei were stained with DAPI (Thermo Fisher; 1:500 in PBS).
  • Z-stacks were taken on a confocal microscope (Leica Stellaris 5), and max projections used to illustrate cell morphology through the scaffolds.
  • Void Space Modeling A custom MATLAB code was used to measure microgel diameters and model annealing. FIJI was used to measure the void space area between microgels.
  • PEG-VS microgels with 1 mM RGD were used for the large and small microgel scaffolds.
  • Microgels were annealed as described above, with the microgel slurry being mixed with heterotypic spheroids before being plated in a 6 mm x 1.5 mm cylindrical silicon mold and exposed to UV light. Scaffolds were collected for analysis on Day 1 and Day 3, with media changed every other day using the 3: 1 mixture of EGM-2:a- MEM.
  • confocal microscopy images cells were stained and imaged with DAPI and phalloidin as described above. Metabolic activity of spheroids was determined by alamarBlue assay (Thermo Fisher). DNA content was quantified using the PicoGreen Quanit-iT Assay Kit (Invitrogen). Migration distance and cell density were quantified in ImageJ using the Li threshold.
  • IC-21 murine macrophages were seeded at 4 million cells mL' 1 in small or large microgel scaffolds.
  • Microgels were formed from 4.5% PEG-VS with 1 mM RGD prepolymer solution.
  • Microgel and macrophage slurries were pipetted into 8 mm x 5 mm cylindrical molds before exposure to UV light.
  • the gels were maintained in RPMI 1640 (ATCC) supplemented with 10% FBS. After 24 hr, gels were moved to a new plate with fresh media and maintained in culture for 6 days with media changes every day.
  • Macrophages with an Ml phenotype were characterized by F4/80+CD86+iNOS+ populations and M2 phenotypes by F4/80+CD206+ARG1+ populations. The frequency of each type of macrophage was quantified per microgel size.
  • Polarization controls consisted of IC-21s seeded on TC wells in monolayer treated with basal media (M0), 200 ng/mL LPS (Ml), and 20 ng/mL IL-4 (M2) for 24 hr. Cells were lifted with trypsin and gentle agitation. Cells were filtered and stained as described.
  • Subfascial Implants Before implantation, 4.5% PEG-VS microgels with 1 mM RGD were loaded and annealed in PDMS molds. Treatment of experimental animals was in accordance with UC Davis animal care guidelines and all National Institutes of Health animal handling procedures. Male twelve-week-old C57BL/6 mice (Jackson Laboratories, West Sacramento, CA) were anesthetized and maintained under a 2% isoflurane/Cb mixture delivered through a nose cone. Each animal received four subfascial implants: small microgels (upper and lower left) and large microgels (upper and lower right). Following a dorsal midline incision, fascia was incised, and blunt dissection was performed between the fascia and muscle belly.
  • Annealed microgels in PDMS molds were placed face-down on the muscle and sutured in place with 4-0 Monocryl sutures (Ethicon, Cornelia, GA). Animals were euthanized after 2 weeks, and gels were collected, removed from PDMS, and fixed in 4% PFA overnight at 4°C. Samples were then washed twice in PBS, paraffin-embedded, and sectioned at 7 pm. Sections were stained with hematoxylin (Thermo) and eosin (Ricca) (H&E) or Masson’s trichrome (Sigma) and imaged using an EVOS XL Core (Invitrogen).

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Abstract

La présente invention concerne des compositions comprenant un échafaudage de microgel formé par du polyéthylèneglycol-vinylsulfone (PEG-VS) et du polyéthylèneglycol-dithiol (PEG-DT), ainsi que des procédés de fabrication correspondants.
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