WO2022154754A1 - Core-shell microspheres - Google Patents

Core-shell microspheres Download PDF

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Publication number
WO2022154754A1
WO2022154754A1 PCT/SG2022/050011 SG2022050011W WO2022154754A1 WO 2022154754 A1 WO2022154754 A1 WO 2022154754A1 SG 2022050011 W SG2022050011 W SG 2022050011W WO 2022154754 A1 WO2022154754 A1 WO 2022154754A1
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cells
gelma
microsphere
shell
core
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French (fr)
Inventor
Lay Poh Tan
Pei Leng TAN
Huizhi CHEN
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Nanyang Technological University
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Nanyang Technological University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5063—Compounds of unknown constitution, e.g. material from plants or animals
    • A61K9/5068—Cell membranes or bacterial membranes enclosing drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5089—Processes
    • 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
    • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
    • A61L26/0061—Use of materials characterised by their function or physical properties
    • A61L26/0066—Medicaments; Biocides
    • 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
    • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
    • A61L26/0095—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • 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
    • A61L27/3804—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 characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney 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
    • A61L27/36—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
    • A61L27/3804—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 characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3813—Epithelial cells, e.g. keratinocytes, urothelial 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
    • A61L27/36—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
    • A61L27/3804—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 characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem 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
    • A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
    • A61L27/48—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with macromolecular fillers
    • 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
    • A61L27/60—Materials for use in artificial skin
    • 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
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
    • A61L2300/62—Encapsulated active agents, e.g. emulsified droplets
    • A61L2300/622—Microcapsules
    • 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
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
    • A61L2300/64—Animal cells

Definitions

  • the present invention generally relates to biotechnology.
  • the present invention relates to a microsphere with a core layer and a shell layer.
  • Microspheres are known to be useful for the delivery of one or more active ingredients.
  • microspheres are made by using synthetic polymers such as polylactic - co-glycolic acid (PLGA) and polylactic acid (PLLA), which allow for the delivery of small molecules for drug therapeutic applications.
  • PLGA polylactic - co-glycolic acid
  • PLLA polylactic acid
  • they have low biocompatibility for cell encapsulation, growth and delivery, therefore have limited use in cell-based and regenerative applications.
  • the degradation rates of these microspheres are uncontrolled, therefore rendering it difficult for the user to control the rate of release of the active ingredients according to their needs.
  • Microspheres are presently fabricated by microfluidics using water-oil emulsions.
  • the use of oil and surfactants limit their translation to the delivery of microspheres into human use.
  • the yield of microspheres from microfluidics are relatively low and the fabrication method often requires multiple steps, which increases the time for fabrication.
  • microfluidics is limited in its versatility as a platform for diverse applications.
  • a microsphere comprising: an inner core layer comprising gelatin methacryloyl (GelMA), wherein the inner core layer further comprises a first cell type; and an outer shell layer comprising gelatin methacryloyl (GelMA) and alginate, wherein the outer shell layer comprises a second cell type.
  • a method of promoting wound healing comprising administering the microsphere as disclosed herein or a synthetic skin replacement as disclosed herein to a subject in need thereof.
  • a method of tissue regeneration comprising administering the microsphere as disclosed herein or a synthetic skin replacement as disclosed herein to a subject in need thereof.
  • a method of treating a disease comprising administering the microsphere as disclosed herein or a synthetic skin replacement as disclosed herein to a subject in need thereof.
  • a method of fabricating the microsphere as disclosed herein comprising: a) preparing an inner core layer solution and an outer shell layer solution; b) setting a core flow rate and a shell flow rate, wherein the sum of the core flow rate and the shell flow rate results in a total flow rate of 9-19 ml/hr; c) electro spraying the microsphere.
  • Figure 1 shows exemplary core-shell microspheres and their uses.
  • A A schematic 3D model of an exemplary core-shell microspheres using GelMA as core and GelMA and alginate as shell.
  • the GelMA in the core can have low degree of substitution (DS) and the GelMA in the shell can have high degree of substitution.
  • B Schematic 3D models of exemplary platforms of core-shell microspheres for encapsulation and co-delivery of (left) two different cell types for cell therapy or (right) two or more different drugs, proteins or nutrients for drug therapeutic application settings.
  • Figure 2 is a schematic illustration of methacrylate substitution with the primary amine of gelatin during gelatin methacryloyl (GelMA) synthesis.
  • Figure 3 is a photo of a cut planar section of fluorescein-labeled core- shell microspheres imaged under a confocal microscope.
  • Figure 4 is a schematic illustration of the experimental set-up of fabricating 3D GelMA core-shell microspheres through co-axial electrospray.
  • FIG. 5 is a schematic illustration of an exemplary experimental set-up of fabricating 3D GelMA core-shell microspheres with HDFs (fibroblasts) and kerCTs (keratinocytes) encapsulated in their respective core-shell compartment through co-axial electro spraying.
  • the same setup can be used for encapsulation of other cells as well as other non-cell components, such as different drugs, proteins or nutrients for different applications such as drug therapeutic applications or cell delivery in regenerative medicine applications.
  • Figure 6 is a 1 H-NMR spectra of pristine gelatin and fabricated GelMA with varying degrees of substitution. Peaks corresponding to (X) acrylic protons (2H) of lysine groups in methacrylamide grafts and those of hydroxyl lysine groups and (Y) methylene protons (2H) of unreacted lysine groups.
  • Figure 9 illustrates the effects of GelMA concentrations and DS on pore size.
  • Figure 10 illustrates the rheological analysis of the storage modulus of GelMA with different DS and different concentration.
  • A A line graph representing the storage modulus of GelMA-DS40 with different concentrations (15%, 10%, 5%).
  • B A line graph representing the storage modulus of GelMA-DS90 with different concentrations (15%, 10%, 5%).
  • Figure 11 illustrates the cell proliferation of cells in GelMA with different DS and different concentration.
  • A A column graph showing the cell proliferation of HDFs in 5%, 10% and 15% (w/v) GelMA-DS40 hydrogels over 7 days.
  • C A column graph showing the cell proliferation of kerCTs in 5%, 10% and 15% (w/v) GelMA-DS90/0.5% alginate hydrogels over 7 days.
  • Figure 16 shows the cell viability of co-cultured kerCTs-HDFs in GelMA core-shell microspheres over 7 days.
  • B Representative fluorescent images of the distribution of live and dead cells in GelMA core-shell microspheres following 3 days of co-culture. Top panel is a gallery view of the z-stack images that show the distribution of live cells.
  • Figure 19 illustrates size distribution of GelMA core-shell microspheres.
  • Figure 20 provides (top) a representative brightfield image showing the morphology of the GelMA core-shell microspheres and (bottom) a column graph showing the size distribution of 200 randomly selected core-shell spheroids.
  • Figure 21 illustrates the yield and coverage analysis of the core-shell microspheres.
  • A (left) A series of photos showing the collection of core-shell microspheres collected in DMEM culture media under 3 minutes of fabrication, (right) The same core-shell microspheres collected in DMEM culture media are displayed on a 9cm dish.
  • B A representative image of a collage of the analysis of fluorescence area covered by FITC-conjugated core-shell microspheres using ImageJ software. Scale was set using the known diameter of the petri dish. The white outline demarcates the area analyzed by ImageJ.
  • Figure 22 illustrates the morphological characterization of cells in GelMA coreshell microspheres
  • A Brightfield images of the morphological characterization of co-cultured skin cells in GelMA core-shell microspheres.
  • (Left to right) Growth of co-cultured HDFs and kerCTs in the core and shell of microspheres (respectively) over 7 days as visualized at (top) 4x magnification and (bottom) lOx magnification.
  • Brightfield imaging was used for day 0 to day 3 while phase contrast imaging was used to better visualize the morphology of released cells from degraded microspheres on day 7.
  • Scalebar 100pm.
  • C Images of analysis of morphological growth of co-cultured HDFs-kerCTs in 3D GelMA core-shell microspheres by immunofluorescence and 3D reconstruction of confocal microscopy images.
  • Figure 23 provides representative phase-contrast images of morphological characterization of delivered mono-cultured kerCTs at (A) 4x, (B) lOx and (C) 20x magnification following 7 days of culture in GelMA core-shell microspheres; and delivered co-cultured HDFs and kerCTs at (D) 4x, (E) lOx and (F) 20x magnification following 7 days of culture in GelMA core-shell microspheres.
  • Figure 24 show representative images of delivered skin cells from GelMA coreshell microspheres after 7 days of co-culture.
  • Figure 25 provide representative images of immunofluorescence staining of growth of delivered kerCTs and encapsulated HDFs in 3D GelMA core-shell microspheres following 7 days of co-culture.
  • Cells were fixed and stained with antibodies against K5 in kerCTs and vimentin in HDFs, counter-stained with Hoechst.
  • A 4x and
  • B lOx magnification of the merged immunofluorescence image.
  • the single channel fluorescence images of (C) Hoechst, (D) K5 and (E) Vimentin staining. Scale bar 100pm.
  • Figure 26 provides images of growth of delivered cells from 3D GelMA core-shell microspheres.
  • Figure 27 provides representative images of magnified fluorescence images depicting the propensity of HDFs to align and elongate along the sheet of kerCTs.
  • Cells were fixed and stained with antibodies against K5 in kerCTs and vimentin in HDFs, counter- stained with Hoechst.
  • the top panel (left to right) represents the phase contrast of delivered cells from GelMA core-shell microspheres before and after washing. A merged immunofluorescence image of all three channels was then presented.
  • the bottom panel (left to right) represents the single channel fluorescence image of Hoechst, K5 and Vimentin staining.
  • Figure 28 provides representative images of fluorescence images depicting the proliferative state of delivered kerCTs and HDFs from 3D GelMA core-shell microspheres following 7 days of co-culture.
  • Cells were fixed and stained with antibodies against ki67 (red) and vimentin in HDFs (green), counter-stained with Hoechst (blue).
  • Phase contrast images of delivered cells from GelMA core-shell microspheres (A) before and (B) after washing.
  • Figure 29 provides representative images of fluorescence images depicting the proliferative state of delivered kerCTs and HDFs from 3D GelMA core-shell microspheres following 14 days of co-culture.
  • Cells were fixed and stained with antibodies against ki67 (red) and vimentin in HDFs (green), counter-stained with Hoechst (blue).
  • Phase contrast images of delivered cells from GelMA core-shell microspheres (A) before and (B) after washing.
  • Figure 32 provides representative images of immunofluorescence staining of growth of fluorescent labelled co-cultured cells (MCF7 cells in core layer and L929 cells in shell layer) over 3 days under different core-shell feed rate.
  • Microspheres are small spherical particles that are commonly used as a delivery platform for active ingredients, for example, drugs, protein, cells, DNA or RNA.
  • Microspheres can have 1 or more layers.
  • a core-shell microsphere has 2 layers, wherein the shell compartment conventionally provides protection of the encapsulated active ingredient against mechanical force during fabrication, enzymatic degradation, or host immune response.
  • most core-shell microspheres are made by synthetic polymers, wherein their low biocompatibility renders them limited in their use for applications such as cell transplantation and regenerative medicine.
  • the inventors have developed a microsphere that can support cell growth and/or have the ability to co-deliver cells and/or active ingredients in a controlled manner.
  • the microsphere of the present disclosure comprises: an inner core layer comprising gelatin methacryloyl (GelMA), wherein the inner core layer further comprises a first cell type; and an outer shell layer comprising gelatin methacryloyl (GelMA) and alginate, wherein the outer shell layer comprises a second cell type.
  • GelMA gelatin methacryloyl
  • the outer shell layer comprises a second cell type.
  • the term “microsphere” refers to a small spherical particle with a diameter of 1-1000 pm.
  • the microsphere has a diameter of, but is not limited to about 100-900 pm, about 200-800 pm, about 300-700 pm, about 400-600 pm, about SOO- SOO pm, or about 100 pm, about 200 pm, about 300 pm, about 400 pm, about 500 pm, about 600 pm, about 700 pm, about 800 pm, about 900 pm, or about 1000 pm.
  • the microsphere has a diameter of about 356-408 pm.
  • the microsphere has a diameter of about 382 pm.
  • the microsphere can be fabricated using organic or inorganic materials, or a combination thereof.
  • the microsphere can comprise an inner core layer and an outer shell layer, wherein the outer shell layer would encapsulate the inner core layer.
  • the outer shell layer confers protection to the inner core layer against physical elements such as mechanical force, or any biological elements such as enzymatic degradation or host immune response, in one example, the outer shell layer is stiffer than the inner core layer.
  • the layers can be distinguished by the materials used to create the outer shell layer and the inner core layer, which would make it possible to determine the thickness of the outer shell layer.
  • the terms “thickness of the outer shell layer” or “shell thickness” refer to the area of the shell and is determined by normalizing the fluorescence area of the outer shell (marked by X in Figure 3) against the planar cross-sectional area of the microsphere (7tr2) where r is the radius of the circle, and calculated using Equation 1 below:
  • the thickness of the shell is important for providing adequate space for the encapsulated cells to grow in the shell, and at the same time it must not be too thick as that could compromise the viability of the cells encapsulated in the core.
  • the thickness of the shell is tunable depending on the applications of the microsphere.
  • the thickness of the outer shell layer can be, but is not limited to about 5-99%, about 10-95%, about 20-95%, about 30-93%, about 40-80%, about 50-70% of the microsphere, or about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 93%, or about 95% of the microsphere. In one example, the thickness of the outer shell layer is about 70% of the microsphere.
  • the inner core layer and/or shell layer of the microsphere can be adjusted to comprise one or more active ingredients, for example, but not limited to cells, drugs, proteins, DNA or RNA.
  • the choice of the active ingredient is dependent on the application that the microsphere is used for.
  • the microsphere can be fabricated to encapsulate cells in the core layer, in the shell layer, or in both the core and shell layers.
  • the microsphere can further comprise one or more cell types.
  • the inner core layer further comprises a first cell type.
  • the outer shell layer further comprises a second cell type.
  • the inner core layer further comprises a first cell type and the outer shell layer further comprises a second cell type.
  • the first cell type in the inner core layer and the second cell type in the outer shell layer can be adjusted based on the requirements of the application in which the microsphere is used for.
  • the first cell type and second cell type comprise anchorage dependent cells, non-anchorage dependent cells, or a combination thereof.
  • anchorage dependent cells refer to one or more cells that can grow, survive, or maintain function only when they are attached to a surface, such as extracellular matrix or tissue in a body, or glass or plastic when culturing the cells in vitro.
  • anchorage dependent cells include, but are not limited to, fibroblasts, keratinocytes, and stem cells such as mesenchymal stem cells.
  • non-anchorage dependent cells refer to one or more cells that can grow, survive, or maintain function when they are not attached to any surface.
  • nonanchorage dependent cells include, but are not limited to cancer stem cells, cancer cells, and hematopoietic cells such as bone marrow mononuclear cells or peripheral blood mononuclear cells.
  • the type of cells that are encapsulated in the core layer and shell layer of the microsphere are dependent on the application that the microsphere is used for.
  • the inner core layer comprises a first cell type selected from a group consisting of fibroblasts, cancer cells, keratinocytes and stem cells.
  • the first cell type is fibroblasts.
  • the outer shell layer comprises a second cell type selected from a group consisting of keratinocytes, fibroblasts, epithelial cells, cancer cells, endothelial cells, and stem cells.
  • the second cell type is keratinocytes.
  • the microsphere can also comprise a combination of the first and second cell type as disclosed herein in the inner core layer and an outer shell layer respectively.
  • the microsphere can further comprise an inner core layer comprising fibroblasts and an outer shell layer comprising keratinocytes.
  • the microsphere can further comprise an inner core layer comprising cancer cells as described herein and an outer shell layer comprising fibroblasts.
  • the microsphere can further comprise an inner core layer comprising breast cancer cells and an outer shell layer comprising fibroblasts.
  • the cell density in the inner core layer and outer shell layer can also be adjusted.
  • the size of microsphere and the thickness of the outer shell layer can be adjusted depending on the cell density in the inner core layer and outer shell layer.
  • the cell density of the inner core layer is, but is not limited to about IxlO 6 cells/ml to 6xl0 6 cells/ml, about 2xl0 6 cells/ml to 5xl0 6 cells/ml, about 3xl0 6 cells/ml to 4xl0 6 cells/ml, or about l.OxlO 6 cells/ml, about 1.5xl0 6 cells/ml, about 2.0xl0 6 cells/ml, about 2.5xl0 6 cells/ml, about 3.0xl0 6 cells/ml, about 3.5xl0 6 cells/ml, about 4.0xl0 6 cells/ml, about 4.5xl0 6 cells/ml, about 5.0xl0 6 cells/ml, about 5.5xl0 6 cells/ml, or about 6.0xl0 6 cells/ml.
  • the inner core layer comprises IxlO 6 cells/ml to 4xl0 6 cells/ml fibroblasts.
  • the inner core layer comprises IxlO 6
  • the cell density of the outer shell layer is, but is not limited to about 5xl0 6 cells/ml to 30xl0 6 cells/ml, about 10xl0 6 cells/ml to 25xl0 6 cells/ml, about 15xl0 6 cells/ml to 20xl0 6 cells/ml, or about 5.0xl0 6 cells/ml, about 6.0xl0 6 cells/ml, about 7.0xl0 6 cells/ml, about 8.0xl0 6 cells/ml, about 9.0xl0 6 cells/ml, about lO.OxlO 6 cells/ml, about l l.OxlO 6 cells/ml, about 12.0xl0 6 cells/ml, about 13.0xl0 6 cells/ml, about 14.0xl0 6 cells/ml, about 15.0xl0 6 cells/ml, about 16.0xl0 6 cells/ml, about 17.0xl0 6 cells/ml, about 18.0xl
  • Gelatin methacryloyl is modified from natural polymer gelatin and retains the tri-amino acid sequence arginine-glycine-aspartic acid (RGD) sequences that gelatin contains to promote cell adhesion.
  • GelMA has a porous micro structure, which provides an optimal environment for encapsulated cells to grow in as it allows diffusion of nutrients oxygen and waste exchange between the culture medium and encapsulated cells.
  • GelMA is also biodegradable, therefore it is used as a material for both the inner core and the outer shell of the microsphere.
  • the biodegradable feature of GelMA provides a time-controlled release of, for example, cells, small molecules or drugs. The time of degradation in either the core or shell layer of the microsphere and the release of, for example, cells can be controlled by adjusting the different parameters of GelMA, for example, the degree of methacryloyl substitution (DS) of GelMA.
  • the terms “degree of substitution”, “degree of methacryloyl substitution” or “DS” refer to the number of substituent groups attached per base unit, in this case, the number of available cross-linkable methacryloyl groups (substituent group) that can crosslink with a photoinitiator crosslinker (base unit), for example, Lithium Phenyl (2,4,6- Trimethylbenzoyl) Phosphinate (LAP) or Irgacure.
  • a photoinitiator crosslinker for example, Lithium Phenyl (2,4,6- Trimethylbenzoyl) Phosphinate (LAP) or Irgacure.
  • photoinitiator refers to a compound that creates reactive species, for example, but not limited to, free radicals, cations or anions when exposed to radiation such as ultraviolet.
  • Photoinitiators are used to initiate a crosslinking or polymerization process upon exposure to radiation.
  • a higher degree of methacryloyation substitution indicates that there is more available cross -linkable methacryloyl groups to crosslink with the photoinitiator crosslinker. This results in a higher crosslinking density after UV irradiation and hence a stiffer hydrogel, which allows it to degrade slower.
  • the degree of methacryloyl substitution is quantified by the 2,4,6- Trinitrobenzenesulfonic acid (TNBSA) method, and calculated using Equation 2 below:
  • the GelMA of the inner core layer comprises a degree of methacryloyl substitution (DS) that can be, but is not limited to about 35%-60%, about 35%- 55%, about 35%-50%, about 40%-55%, about 45%-50%, in particular about 38%-46%.
  • DS degree of methacryloyl substitution
  • the GelMA of the inner core layer comprises a degree of methacryloyl substitution (DS) that can be, but is not limited to about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, or about 60%.
  • the DS of the GelMA of the inner core layer is about 41.73%.
  • the GelMA of the outer shell layer comprises a degree of methacryloyl substitution (DS) that can be, but is not limited to about 65%-96%, about 75%- 95%, about 85%-95%, in particular about 86%-95%.
  • DS degree of methacryloyl substitution
  • the GelMA of the outer shell layer comprises a degree of methacryloyl substitution (DS) that can be, but is not limited to about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,.
  • the DS of the GelMA of the outer shell layer is about 90.74%.
  • the different DS can be rounded to their nearest tens for ease of terminology.
  • the DS of GelMA that is 41.73%, 57.10% and 90.74% can be labeled as GelMA- DS40, GelMA-DS60 and GelMA-DS90 respectively.
  • GelMA-DS90 is stiffer than the GelMA - DS40. Stiffness can be measured by the storage modulus.
  • the term “storage modulus” refers to the measure of the amount of energy required to be put into a material in order to distort it. Storage modulus provides an indication of the stiffness of a solid material. A higher storage modulus indicates a stiffer material.
  • the inner core layer comprises a storage modulus of 1.30 Pa to 4.61 kPa.
  • the inner core layer comprises a storage modulus that can be, but is not limited to about 50-500 Pa, about 500 Pa- 1 kPa, about 1-1.5 kPa, about 1.5-2 kPa, about 2-2.5 kPa, about 2.5-3 kPa, about 3-3.5 kPa, about 3.5-4 kPa, about 4-4.5 kPa, or about 500 Pa, about 1 kPa, about 1.5 kPa, about 2 kPa, about 2.5 kPa, about 3 kPa, about 3.5 kPa, about 4 kPa, or about 4.5 kPa.
  • the inner core layer has a storage modulus of about 4.61 kPa.
  • the inner core layer has a storage modulus of about 821 Pa.
  • the outer shell layer should have a higher storage modulus.
  • the outer shell layer comprises a storage modulus of 14.96 Pa to 13.3 kPa.
  • the inner core layer comprises a storage modulus that can be, but is not limited to about 50-500 Pa, about 500 Pa-1 kPa, about 1-1.5 kPa, about 1.5-2 kPa, about 2-2.5 kPa, about 2.5-3 kPa, about 3-3.5 kPa, about 3.5-4 kPa, about 4-4.5 kPa, about 4.5-5 kPa, about 5-5.5 kPa, about 5.5-6 kPa, about 6-6.5 kPa, about 6.5-7 kPa, about 7-7.5 kPa, about 7.5-8 kPa, about 8-8.5 kPa, about 8.5-9 kPa, about 9-9.5 kPa, about 9.5- 10 kPa, about 10-10.5 k
  • the time of degradation and the release of, for example, cells can also be controlled by adjusting the concentration of GelMA.
  • the inner core layer and outer shell layer each comprise a concentration of about 5-15% (w/v) GelMA.
  • the inner core layer and outer shell layer each comprise a concentration of, but is not limited to about 5% (w/v), about 6% (w/v), about 7% (w/v), about 8% (w/v), about 9% (w/v), about 10% (w/v), about 11% (w/v), about 12% (w/v), about 13% (w/v), about 14% (w/v), or about 15% (w/v).
  • the inner core layer and outer shell layer each have a concentration of about 10% (w/v) GelMA.
  • the outer shell layer of the micro sphere further comprises alginate.
  • the addition and concentration of alginate in GelMA can also control the time of degradation and the release of, for example, cells.
  • the outer shell layer comprises about 0.1- 1.0% alginate. In another example, the outer shell layer comprises about 0.5% alginate.
  • the combination of the degree of methacryloyl substitution (DS) and concentration of GelMA can affect the pore size of the inner core layer and outer shell layer of the microsphere.
  • the pores in the inner core layer and outer shell layer of the microsphere influence the adhesion of cells while facilitating the diffusion of nutrients, oxygen, and waste exchange to the encapsulated cells. Having pore size that is too small can reduce the efficiency of diffusion of nutrients, oxygen, and waste exchange to the encapsulated cells. On the other hand, if the pore size is too large, the cells might not be able to adhere properly.
  • the inner core layer comprises a pore size of, but is not limited to, about 10 - 70 pm, about 20 - 60 pm, about 30 - 50 pm, about 35 - 45 pm, or about 20 pm, about 21 pm, about 22 pm, about 23 pm, about 24 pm, about 25 pm, about 26 pm, about 27 pm, about 28 pm, about 29 pm, about 30 pm, about 31 pm, about 32 jam, about 33
  • the outer shell layer comprises a pore size of, but is not limited to, about 20 - 50 pm, about 25 - 45 pm, about 30 - 40 pm, or about 20 pm, about 21 pm, about 22 pm, about 23 pm, about 24 pm, about 25 pm, about 26 pm, about 27 pm, about 28 pm, about 29 pm, about 30 pm, about 31 pm, about 32 pm, about 33 pm, about 34 pm, about 35 pm, about 36 pm, about 37 pm, about 38 pm, about 39 pm, about 40 pm, about 41 pm, about 42 pm, about 43 pm, about 44 pm, about 45 pm, about 46 pm, about 47 pm, about 48 pm, about 49 pm, or about 50 pm.
  • the outer shell layer comprises a pore size of about 27 - 39 pm.
  • the outer shell layer pore size is about 36.83 pm.
  • the microsphere as disclosed herein is tunable by combining different parameters as disclosed herein to meet the requirements of the application in which the microsphere is used for.
  • the microsphere can be cell-free.
  • the microsphere comprises: an inner core layer comprising 5-15% (w/v) gelatin methacryloyl (GelMA) and a storage modulus of 1.30 Pa to 4.61 kPa; an outer shell layer comprising 5-15% (w/v) gelatin methacryloyl (GelMA), a storage modulus of 14.96 Pa to 13.3 kPa, and alginate; wherein the thickness of the outer shell layer is about 5-99% of the microsphere.
  • the microsphere comprises: an inner core layer comprising 5-15% (w/v) gelatin methacryloyl (GelMA) and a pore size of 13-65 pM; an outer shell layer comprising 5-15% (w/v) gelatin methacryloyl (GelMA), a pore size of 27-39 pM, and alginate; wherein the thickness of the outer shell layer is about 5-99% of the microsphere.
  • the microsphere comprises: an inner core layer comprising 5-15% (w/v) gelatin methacryloyl (GelMA) with a degree of methacryloyl substitution (DS) of about 30-60%; an outer shell layer comprising 5-15% (w/v) gelatin methacryloyl (GelMA) with a degree of methacryloyl substitution (DS) of about 65-96% and alginate; wherein the thickness of the outer shell layer is about 5-99% of the microsphere.
  • the microspheres as disclosed herein can encapsulate one or more types of cells in the core and/or shell layer.
  • the microsphere comprises: an inner core layer comprising 5-15% (w/v) gelatin methacryloyl (GelMA), a storage modulus of 1.30 Pa to 4.61 kPa, and a first cell type; an outer shell layer comprising 5-15% (w/v) gelatin methacryloyl (GelMA), a storage modulus of 14.96 Pa to 13.3 kPa, and alginate; wherein the thickness of the outer shell layer is about 5-99% of the microsphere.
  • the microsphere comprises: an inner core layer comprising 5-15% (w/v) gelatin methacryloyl (GelMA), a pore size of 13-65 pM, and a first cell type; an outer shell layer comprising 5-15% (w/v) gelatin methacryloyl (GelMA), a pore size of 27-39 pM, and alginate; wherein the thickness of the outer shell layer is about 5-99% of the microsphere.
  • the microsphere comprises: an inner core layer comprising 5-15% (w/v) gelatin methacryloyl (GelMA) with a degree of methacryloyl substitution (DS) of about 30-60% and and a first cell type; an outer shell layer comprising 5-15% (w/v) gelatin methacryloyl (GelMA) with a degree of methacryloyl substitution (DS) of about 65-96% and alginate; wherein the thickness of the outer shell layer is about 5-99% of the microsphere.
  • GelMA gelatin methacryloyl
  • DS degree of methacryloyl substitution
  • the microsphere comprises: an inner core layer comprising 5-15% (w/v) gelatin methacryloyl (GelMA) and a storage modulus of 1.30 Pa to 4.61 kPa; an outer shell layer comprising 5-15% (w/v) gelatin methacryloyl (GelMA), a storage modulus of 14.96 Pa to 13.3 kPa, alginate and a second cell type; wherein the thickness of the outer shell layer is about 5-99% of the microsphere.
  • the microsphere comprises: an inner core layer comprising 5-15% (w/v) gelatin methacryloyl (GelMA) and a pore size of 13-65 pM; an outer shell layer comprising 5- 15% (w/v) gelatin methacryloyl (GelMA), a pore size of 27-39 pM, alginate, and a second cell type; wherein the thickness of the outer shell layer is about 5-99% of the microsphere.
  • the microsphere comprises: an inner core layer comprising 5-15% (w/v) gelatin methacryloyl (GelMA) with a degree of methacryloyl substitution (DS) of about 30- 60%; an outer shell layer comprising 5-15% (w/v) gelatin methacryloyl (GelMA) with a degree of methacryloyl substitution (DS) of about 65-96%, alginate, and a second cell type; wherein the thickness of the outer shell layer is about 5-99% of the microsphere.
  • an inner core layer comprising 5-15% (w/v) gelatin methacryloyl (GelMA) with a degree of methacryloyl substitution (DS) of about 30- 60%
  • an outer shell layer comprising 5-15% (w/v) gelatin methacryloyl (GelMA) with a degree of methacryloyl substitution (DS) of about 65-96%, alginate, and a second cell type
  • the thickness of the outer shell layer is about 5-99%
  • the microsphere comprises: an inner core layer comprising 5-15% (w/v) gelatin methacryloyl (GelMA), a storage modulus of 1.30 Pa to 4.61 kPa and a first cell type; an outer shell layer comprising 5-15% (w/v) gelatin methacryloyl (GelMA), a storage modulus of 14.96 kPa to 13.3 kPa, alginate and a second cell type; wherein the thickness of the outer shell layer is about 5-99% of the microsphere.
  • the microsphere comprises: an inner core layer comprising 5-15% (w/v) gelatin methacryloyl (GelMA), a pore size of 13-65 pM and a first cell type; an outer shell layer comprising 5-15% (w/v) gelatin methacryloyl (GelMA), a pore size of 27-39 pM, alginate and a second cell type; wherein the thickness of the outer shell layer is about 5-99% of the microsphere.
  • the microsphere comprises: an inner core layer comprising 5-15% (w/v) gelatin methacryloyl (GelMA) with a degree of methacryloyl substitution (DS) of about 30-60% and a first cell type; and an outer shell layer comprising 5-15% (w/v) gelatin methacryloyl (GelMA) a degree of methacryloyl substitution (DS) of about 65-96%, alginate, wherein the thickness of the outer shell layer is about 5-99% of the microsphere.
  • GelMA gelatin methacryloyl
  • DS degree of methacryloyl substitution
  • the microsphere comprises: an inner core layer comprising 5-15% (w/v) gelatin methacryloyl (GelMA) with a degree of methacryloyl substitution (DS) of about 30-60% and a first cell type; and an outer shell layer comprising 5-15% (w/v) gelatin methacryloyl (GelMA) a degree of methacryloyl substitution (DS) of about 65-96%, 0.1-1.0% alginate, and a second cell type, wherein the thickness of the outer shell layer is about 5-99% of the microsphere.
  • GelMA gelatin methacryloyl
  • DS degree of methacryloyl substitution
  • the microsphere comprises: an inner core layer comprising 10% (w/v) gelatin methacryloyl (GelMA) with a degree of methacryloyl substitution (DS) of about 41.73% and a first cell type; and an outer shell layer comprising 10% (w/v) gelatin methacryloyl (GelMA) a degree of methacryloyl substitution (DS) of about 90.74%, 0.5% alginate, and a second cell type, wherein the thickness of the outer shell layer is about 70% of the microsphere.
  • GelMA gelatin methacryloyl
  • DS degree of methacryloyl substitution
  • the microsphere comprises: an inner core layer comprising 10% (w/v) gelatin methacryloyl (GelMA) with a degree of methacryloyl substitution (DS) of about 41.73% and fibroblasts; and an outer shell layer comprising 10% (w/v) gelatin methacryloyl (GelMA) a degree of methacryloyl substitution (DS) of about 90.74%, 0.5% alginate, and keratinocytes, wherein the thickness of the outer shell layer is about 70% of the microsphere.
  • GelMA gelatin methacryloyl
  • DS degree of methacryloyl substitution
  • the microspheres as disclosed herein have tunable properties for different applications by fine-tuning the different hydrogel formulations that are used for both core and shell compartment.
  • By tailoring the formulations of the inner core and outer shell layers of the microsphere as described herein different mechanical strength and degradation rates can be achieved, resulting in a tunable diffusion and release time of co-encapsulated active ingredients or cells according to the needs of applications.
  • the application can be, but is not limited to manufacturing therapeutic products, delivery of a 2D or 3D co-cultured cell in a biomedical setting, therapeutic drug or small molecules delivery, and food science.
  • a synthetic skin replacement that is manufactured from the microsphere as disclosed herein.
  • Another example of the application is the method of manufacturing a synthetic skin replacement using the microsphere as disclosed herein.
  • the synthetic skin replacement is selected from a group consisting of an epidermal-dermal skin sheet, an epidermal skin sheet and a dermal skin sheet.
  • the microsphere, synthetic skin replacement or composition as disclosed herein can be used in a clinical setting and be used to treat a subject in need thereof.
  • a method of promoting wound healing comprising administering the microsphere as disclosed herein or a synthetic skin replacement as disclosed herein to a subject in need thereof.
  • the microsphere as disclosed herein or a synthetic skin replacement as disclosed herein is for use in promoting wound healing.
  • a method of tissue regeneration comprising administering the microsphere as disclosed herein or a synthetic skin replacement as disclosed herein to a subject in need thereof.
  • the microsphere as disclosed herein or a synthetic skin replacement as disclosed herein is for use in tissue regeneration.
  • a method of treating a disease comprising administering the microsphere as disclosed herein or a synthetic skin replacement as disclosed herein in a subject in need thereof.
  • the microsphere as disclosed herein or a synthetic skin replacement as disclosed herein is for use in treating a disease.
  • the use of the microsphere as disclosed herein or a synthetic skin replacement as disclosed herein in the manufacture of a medicament for treating a disease can be a skin disease or disorder, wherein the skin disease or disorder comprises bum injury, Recessive Dystrophic Epidermolysis Bullosa, diabetic foot ulcers, infectious wounds, ischemic wounds, open wounds and chronic wound.
  • the administration comprises topical, subcutaneous, intravenous, or intramuscular administration.
  • the method Prior to treating a subject in need thereof, the method can further comprise pretreating the microsphere as disclosed herein with trypsin before administration.
  • the trypsin can be any trypsin that is commonly used, for example, 0.25% trypsin EDTA.
  • the microsphere can be pre-treated for at least 5 minutes or for about 5 minutes.
  • the method of fabricating the microsphere has high tunability, high yield, high scalability and high encapsulation efficiency.
  • yield refers to the percentage of nondefective items of all produced items, as indicated by the ratio of the number of non-defective items against the number of manufactured items.
  • 100% of all the materials used can be directly sprayed into microspheres, unlike in other fabrication process where some of the materials are lost due to external environment (heat, stirring), or through chemical reaction means.
  • the present invention discloses a method of fabricating the microsphere that can have 100% yield microspheres, as well as the fabrication of a large amount of microspheres in a short amount of time.
  • the method of fabricating the microsphere as disclosed herein comprises: a) preparing an inner core layer solution and an outer shell layer solution; b) setting a core flow rate and a shell flow rate, wherein the sum of the core flow rate and the shell flow rate results in a total flow rate of 9-19 ml/hr; c) electro spraying the microsphere.
  • the shell thickness of the core-shell microspheres can be facilely tuned by changing the flow rate of core and shell hydrogel solutions, thereby tuning the release of encapsulated core ingredients.
  • the total flow rate 15 ml/hr.
  • the core flow rate is about 1- 14 ml/hr, and it would be apparent that the shell flow rate is about 14-1 ml/hr.
  • the following combination can be, but is not limited to: the core flow rate is about 1 ml/hr and the shell flow rate is about 14 ml/hr, the core flow rate is about 2 ml/hr and the shell flow rate is about 13 ml/hr, the core flow rate is about 3 ml/hr and the shell flow rate is about 12 ml/hr, the core flow rate is about 4 ml/hr and the shell flow rate is about 11 ml/hr, the core flow rate is about 5 ml/hr and the shell flow rate is about 10 ml/hr, the core flow rate is about 6 ml/hr and the shell flow rate is about 9 ml/hr, the core flow rate is about 7 ml/hr
  • the inner core layer solution comprises gelatin methacryloyl (GelMA) as disclosed herein.
  • the GelMA of the inner core layer solution comprises a degree of methacryloyl substitution (DS) of about 35%-60%.
  • the DS of the GelMA of the inner core layer solution is about 41.73%.
  • the outer shell layer solution comprises gelatin methacryloyl (GelMA) and alginate as disclosed herein.
  • the GelMA of the outer shell layer solution comprises a degree of methacryloyl substitution (DS) of about 65%-96%.
  • the DS of the GelMA of the outer shell layer solution is about 90.74%.
  • the outer shell layer solution comprises about 0.1- 1.0% alginate. In another example, the outer shell layer solution comprises about 0.5% alginate.
  • the inner core layer solution and outer shell layer solution each further comprise a photoinitiator.
  • the photoinitiator comprises Lithium Phenyl (2,4,6- Trimethylbenzoyl) Phosphinate (LAP) or Irgacure.
  • the photoinitiator is Lithium Phenyl (2,4,6-Trimethylbenzoyl) Phosphinate (LAP).
  • the photoinitiator is 0.1 % LAP.
  • the inner core layer solution and outer shell layer solution each comprise a concentration of about 5-15% (w/v) GelMA.
  • the inner core layer and outer shell layer each comprise a concentration of, but is not limited to about 5% (w/v), about 6% (w/v), about 7% (w/v), about 8% (w/v), about 9% (w/v), about 10% (w/v), about 11% (w/v), about 12% (w/v), about 13% (w/v), about 14% (w/v), or about 15% (w/v).
  • the inner core layer and outer shell layer each have a concentration of about 10% (w/v) GelMA.
  • the inner core layer solution can further comprise a first cell type.
  • the outer shell solution can further comprise a second cell type.
  • the first cell type in the inner core layer solution and the second cell type in the outer shell layer solution can be adjusted based on the requirements of the application in which the microsphere is used for.
  • the first cell type and second cell type comprise anchorage dependent cells as disclosed herein, non-anchorage dependent cells as disclosed herein, or a combination thereof.
  • the first cell type is selected from a group consisting of fibroblasts, cancer cells, keratinocytes and stem cells.
  • the first cell type is fibroblasts.
  • the second cell type is selected from a group consisting of keratinocytes, fibroblasts, epithelial cells, cancer cells, endothelial cells, and stem cells. In another example, the second cell type is keratinocytes.
  • the cell density of in the inner core layer solution and outer shell layer solution can also be adjusted.
  • the cell density of the inner core layer solution is, but is not limited to about IxlO 6 cells/ml to 6xl0 6 cells/ml, about 2xl0 6 cells/ml to 5xl0 6 cells/ml, about 3xl0 6 cells/ml to 4xl0 6 cells/ml, or about l.OxlO 6 cells/ml, about 1.5xl0 6 cells/ml, about 2.0xl0 6 cells/ml, about 2.5xl0 6 cells/ml, about 3.0xl0 6 cells/ml, about 3.5xl0 6 cells/ml, about 4.0xl0 6 cells/ml, about 4.5xl0 6 cells/ml, about 5.0xl0 6 cells/ml, about 5.5xl0 6 cells/ml, or about 6.0xl0 6 cells/ml.
  • the inner core layer solution comprises a cell density of IxlO 6 cells/ml to 4xl0 6 cells/ml fibroblasts. In another example, the inner core layer solution comprises a cell density of 4xl0 6 cells/ml fibroblasts.
  • the cell density of the outer shell layer solution is, but is not limited to about 5xl0 6 cells/ml to 30xl0 6 cells/ml, about 10xl0 6 cells/ml to 25xl0 6 cells/ml, about 15xl0 6 cells/ml to 20xl0 6 cells/ml, or about 5.0xl0 6 cells/ml, about 6.0xl0 6 cells/ml, about 7.0xl0 6 cells/ml, about 8.0xl0 6 cells/ml, about 9.0xl0 6 cells/ml, about lO.OxlO 6 cells/ml, about l l.OxlO 6 cells/ml, about 12.0xl0 6 cells/ml, about 13.0xl0 6 cells/ml, about 14.0xl0 6 cells/ml, about 15.0xl0 6 cells/ml, about 16.0xl0 6 cells/ml, about 17.0xl0 6 cells/ml, about 18.0x
  • the outer shell layer solution comprises a cell density of 5xl0 6 cells/ml to 20xl0 6 cells/ml keratinocytes. In another example, the outer shell layer solution comprises a cell density of 20xl0 6 cells/ml keratinocytes.
  • Step c) of the method of fabricating the microsphere as disclosed herein can comprise a co-axial nozzle for electro spraying.
  • the co-axial nozzle is 16-21G or 18-14G.
  • an applied voltage needs to be set for electro spraying.
  • the applied voltage can be, but is not limited to, about 7.5 - 12kV, about 8 - l lkV, about 9 - lOkV, or about 7.5kV, about 8.0kV, about 8.5kV, about 9.0kV, about 9.5kV, about lO.OkV, about 10.5kV, or about 1 l.OkV.
  • the applied voltage is about 9kV.
  • the method of fabricating the microsphere further comprises d) collecting the microsphere in BaCh or CaCh.
  • the concentration of BaCh or CaCh can be about 50mM to about 150mM.
  • the concentration of BaCh or CaCh can be, but is not limited to about 50mM, about 60mM, about 70mM, about 80mM, about 90mM, about lOOmM, about l lOmM, about 120mM, about 130mM, about 140mM or about 150mM. In another example, the concentration of BaCh or CaCh is lOOmM.
  • the method of fabricating the microsphere further comprises e) exposing the microsphere from step d) to ultraviolet.
  • Figure 4 provides an exemplary method as disclosed herein, wherein the method of fabricating the microsphere comprises: a) preparing an inner core layer solution and an outer shell layer solution; b) setting a core flow rate and a shell flow rate, wherein the sum of the core flow rate and the shell flow rate results in a total flow rate; c) electro spraying the microsphere using a set applied voltage; d) collecting the microsphere in a collector bath; and e) exposing the microsphere from step d) to ultraviolet.
  • Figure 5 provides another exemplary method as disclosed herein, wherein the method of fabricating the microsphere comprises: a) preparing an inner core layer solution comprising a first cell type, for example, fibroblasts, and an outer shell layer solution comprising a second cell type, for example, keratinocytes; b) setting a core flow rate and a shell flow rate, wherein the sum of the core flow rate and the shell flow rate results in a total flow rate; c) electro spraying the microsphere using an applied voltage; d) collecting the microsphere in (e.g. lOOmM) BaCh; and e) exposing the microsphere from step d) to ultraviolet.
  • a) preparing an inner core layer solution comprising a first cell type, for example, fibroblasts, and an outer shell layer solution comprising a second cell type, for example, keratinocytes b) setting a core flow rate and a shell flow rate, wherein the sum of the core flow rate and the shell flow rate results in a total flow rate;
  • the cell encapsulated microspheres are collected, washed and transferred into cell culture.
  • a genetic marker includes a plurality of genetic markers, including mixtures and combinations thereof.
  • the terms “increase” and “decrease” refer to the relative alteration of a chosen trait or characteristic in a subset of a population in comparison to the same trait or characteristic as present in the whole population. An increase thus indicates a change on a positive scale, whereas a decrease indicates a change on a negative scale.
  • the term “change”, as used herein, also refers to the difference between a chosen trait or characteristic of an isolated population subset in comparison to the same trait or characteristic in the population as a whole. However, this term is without valuation of the difference seen.
  • the term “about” in the context of concentration of a substance, size of a substance, length of time, or other stated values means +/- 5% of the stated value, or +/- 4% of the stated value, or +/- 3% of the stated value, or +/- 2% of the stated value, or +/- 1% of the stated value, or +/- 0.5% of the stated value.
  • range format may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • Type A GelMA samples with three different degrees of substitution were first synthesized. 7.95g of Na2COs and 14.65g of NaHCCh were dissolved in IL of distilled water to prepare 0.25M of carbonate-bicarbonate (CB) buffer solution. Following that, 30g of type A gelatin from porcine skin (gel strength ⁇ 175g Bloom, Sigma- Aldrich, St. Louis, MO, USA) was dissolved in 300 mL of the as-prepared 0.25M CB buffer at 50°C. After the gelatin is homogeneously mixed in the CB buffer, the pH value of the gelatin solution was adjusted to 9.
  • CB carbonate-bicarbonate
  • anhydrous methacrylic anhydride (94%, Sigma) were separately added to the gelatin solution at a MAA (mL) /gelatin (g) ratio of 0.05, 0.063 and 0.1 mL/g for a target of low, moderate and high DS GelMA under magnetic stirring at 500 rpm.
  • the reaction was left to proceed at 50°C for 3 hours.
  • the reaction was left to proceed without any adjustment of pH whereas for a targeted moderate and high DS GelMA, the pH value of gelatin solution was adjusted to 9 after 30 minutes of MAA addition.
  • IM HC1 was added to the solution and the reaction was stopped when the pH of the solution was reduced to 7.4.
  • the solution was then filtered and dialyzed against DI water at 50°C in a 14kDa dialysis membrane (Membra-CelTM) to remove any unreacted MA and methacrylic acid by-product.
  • the GelMA solution was lyophilized until a dried solid product was obtained and stored at -20°C for future use.
  • the frequency of the time-sweep test was set at 1 Hz with a constant shear strain of 3% throughout the entire 6 minutes of the test. After 60s of starting the time- sweep test, the GelMA pre-polymer solutions were irradiated with UV and were crosslinked for 5 minutes before the test stops.
  • HDFs were maintained in Dulbecco's modified Eagle's medium (DMEM, Life Technologies) supplemented with 10% fetal bovine serum (FBS, Life Technologies) and 1% of penicillin (100 units/mL) and streptomycin (100 pg/mL) (Life Technologies).
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • penicillin 100 units/mL
  • streptomycin 100 pg/mL
  • kerCTs were maintained in serum-free conditions in keratinocyte basal medium gold (KBM-Gold) (Lonza, Basel, Switzerland) supplemented with KGM-Gold SingleQuots (Lonza) comprising of individual vials of hydrocortisone, transferrin, epinephrine, gentamicin sulfate/amphotericin-B (GA- 1000), bovine pituitary extract, human epidermal growth factor and insulin.
  • KBM-Gold keratinocyte basal medium gold
  • G- 1000 gentamicin sulfate/amphotericin-B
  • bovine pituitary extract bovine pituitary extract
  • human epidermal growth factor and insulin The cells were cultured in T150 tissue culture flasks (Corning®, New York, USA) in a 37°C incubator with 5% CO2, with subsequent change in media every 2 days until approximately 80% confluent. Cells passaged 5-9 times were selected for all cell studies.
  • MCF7 breast cancer cells and L929 fibroblast cells were maintained in a Dulbecco's modified Eagle's medium (DMEM, Life Technologies) supplemented with 10% fetal bovine serum (FBS, Life Technologies) and 1% of penicillin (100 units/mL) and streptomycin (100 pg/mL) (Life Technologies) at 37°C in the presence of 5% CO2.
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • penicillin 100 units/mL
  • streptomycin 100 pg/mL
  • GelMA-DS40 and GelMA-DS90 macromers were dissolved in phosphate-buffered saline (PBS, Life Technologies) to achieve a stock concentration of 20% (w/v) GelMA-DS40 solution and 25% (w/v) GelMA-DS90 solution respectively.
  • PBS phosphate-buffered saline
  • a stock sodium alginate solution was also prepared by dissolving 2% (w/v) sodium alginate (NF, Spectrum Chemical Manufacturing Corp., New Brunswick, USA) in Milli-Q water. The stock solutions were then sterile-filtered through Minisart® polyethersulfone 0.22pm syringe filter (Sartorius, Gottingen, Germany).
  • a fixed volume of cell suspension and 2% sodium alginate (for GelMA/alginate hydrogels) was then mixed with a varying volume of PBS and GelMA stock solutions to achieve a final GelMA concentration of 10% GelMA-DS25 with 10x106 cells/mL MCF-7 cells as the core solution and 10% GelMA-DS96/0.5% alginate with cell concentration of 5x106 cells/mL of L929 cells as the shell solution.
  • the photoinitiator, LAP was also added to the core and shell solutions to make up into a final concentration 0.1%.
  • MCF-7 and L929 cells were stained with cells tracker dye DiO (red) and Dil (green) respectively at a concentration of 1:400 (v/v) in PBS and incubated at 37°C for an hour. The fluorescent-tagged cells were then collected by centrifuging at 1500 rpm for 5 minutes before adding to the core and shell GelMA solutions respectively.
  • the core and shell solutions were electro -sprayed into a lOOmM BaC12 collector bath and UV crosslinked for 5mins. Subsequently, the cell-laden core-shell microspheres were rinsed in sodium chloride solution before transferring to a 12-well plate containing DMEM media supplemented with 10% FBS and 1% antibiotic-antimycotic solution.
  • a standard curve correlating known cell numbers to the absorbance value of CCK- 8 at 450nm was first done.
  • HDFs were seeded in a range of IxlO 5 cells to 5xl0 5 cells in 10% GelMA-DS40 hydrogels whereas kerCTs were seeded in a range of IxlO 5 cells to 5xl0 5 cells in 10% GelMA-DS90/0.5% alginate hydrogels.
  • the cell-laden hydrogels were cultured in 200pL of their respective culture medium for 24 hours before replacing with 10% (v/v) CCK- 8 in HDFs or kerCTs culture medium. The samples were then incubated at 37°C for 2 hours in the dark.
  • a net absorbance value of cells in each concentration of GelMA hydrogel was obtained by deducting the obtained absorbance value of each cell-laden hydrogel sample with that of its blank hydrogel. The actual cell number was then quantified from the net absorbance value using the linear fit equations obtained from standard curves.
  • Fluorescein-labeled alginate was synthesized by conjugating sodium alginate (Spectrum Chemical Manufacturing Corp.) with fluoresceinamine (isomer I, Sigma) via an EDC-NHS coupling reaction. [00130] Briefly, 150 mg of sodium alginate was dissolved in 10.0 mL PBS overnight. Following that, 7.5 mg of fluoresceinamine, 450 mg of N-(3-Dimethylaminopropyl)-N’- ethylcarbodiimide hydrochloride (EDC, Sigma) and 225 mg of N-Hydroxysuccinimide (NHS, Sigma) were added into the alginate solution. The reaction was left to proceed at room temperature for 24 hours under stirring.
  • EDC N-(3-Dimethylaminopropyl)-N’- ethylcarbodiimide hydrochloride
  • NHS N-Hydroxysuccinimide
  • the solution was then dialyzed against water in a 3500 Da dialysis membrane for 5 days and the water was replaced 2-3 times per day. Finally, the fluorescein-conjugated alginate solution was lyophilized until a dried solid product was obtained and stored in a vacuum desiccator for further use.
  • the shell thickness of the microspheres fabricated under varying coreshell flow rates was captured using a confocal microscope. Through the planar fluorescent images, the shell thickness was determined by normalizing the fluorescence area of the shell (marked by X in Figure 3) against the planar cross-sectional area of the microsphere (7tr2) where r is the radius of the circle. The radius was quantified based on the diameter of the microspheres, as measured using ImageJ software (line Y in Figure 3).
  • Table 1 List of primary antibodies diluted in blocking buffer at optimized concentration used in the various studies.
  • cells were washed with PBS thrice, for 5 minutes each. Thereafter, fluorochrome-conjugated secondary antibodies and a nuclear staining dye, Hoechst 33342, were added to the samples and left at room temperature for an hour.
  • the fluorochrome-conjugated secondary antibodies targeting mouse and rabbit antibodies were diluted in the blocking buffer at the following concentrations listed in Table 2.
  • Hoechst 33342 dye was also added to the same blocking buffer solution at a concentration of 1:1000.
  • the immunofluorescence- stained cells were washed with PBS thrice for 5 minutes each, before analyzing under a fluorescence microscope.
  • Keratinocytes and fibroblasts are of different origins and are characterized according to their specific phenotypic markers.
  • Cytokeratin-5 (K5), a type II intermediate filament protein, is primarily expressed in basal keratinocytes of the epithelial cells while vimentin, a type III intermediate filament, constitute the major cytoskeletal component of mesenchymal cells such as fibroblasts.
  • kerCTs and HDFs were co-cultured over 7 days in serum-free co-culture media and the media was changed every 2 days.
  • the cell viability of kerCTs-HDFs encapsulated in 3D GelMA core-shell microspheres was studied using Live/Dead® Viability Assay Kit (Life Technologies) consisting of 4 mM Calcein AM and 2 mM Ethidium homodimer- 1 (EthD-1).
  • a live-dead solution was first prepared by diluting the Calcein AM and EthD-1 to a final concentration of 2pM and 4pM respectively in a serum-free co-culture media.
  • kerCTs and HDFs encapsulated in GelMA core-shell microspheres were co-cultured over 7 days in serum-free co-culture media and the media was changed every 2 days.
  • the microspheres-containing cell strainers were first removed from the well plate at each time point and placed into a new 6-well plate. The strainers were inverted and a fixed volume of the live-dead staining solution was pipetted over the strainers to transfer the cell-laden microspheres into the new well plate. The microspheres were then incubated in the live-dead solution for 30 minutes at 37°C. This step is carried out to reduce the amount of live-dead solution required, as staining the cell-laden microspheres along with the large cell strainers would require about 5 mL of live-dead solution for each sample.
  • the stained cellladen microspheres were filtered and collected using a new cell strainer.
  • the cell strainer now containing stained cell-laden microspheres were thoroughly washed with pre-warmed PBS three times and subsequently placed into a well plate containing 0.25% Trypsin-EDTA with enough volume ( ⁇ 5 mL) to fully cover the microspheres.
  • the well plate with strainers was then incubated at 37°C for 5 minutes for the digestion of microspheres to take place. Thereafter, the digestion was stopped by adding a complete medium to the well plate and collecting the digested cell suspension into a 15 mL centrifuge tube. A cell pellet was obtained after centrifuging at 1200 rpm for 5 minutes. The supernatant was aspirated and the cell pellet was gently washed with PBS before centrifuging the cell suspension.
  • the cell viability was then quantified by normalizing the number of live cells by the total number of cells and expressed as a cell viability percentage based on Equation 4.
  • the proliferation of kerCTs in monoculture and co-culture systems was then investigated following 1, 3, 5 and 7 days of culture.
  • the cell strainers containing cell-laden microspheres were removed from the well plate at each time point and washed twice with pre-warmed PBS. The strainers were subsequently placed into a well plate containing 0.25% Trypsin-EDTA with enough volume to fully cover the microspheres and incubated at 37 °C for 5 minutes for the microspheres to be digested. Thereafter, the digestion was stopped by adding a complete medium to the well plate and collecting the digested cell suspension into a 15 mL centrifuge tube. A cell pellet was obtained after centrifuging at 1200 rpm for 5 minutes.
  • the supernatant was aspirated and the cell pellet was gently resuspended with PBS before transferring to a 2 mL Eppendorf tube.
  • the Eppendorf tubes were then centrifuged at 15x100 g for 5 minutes in a microcentrifuge (PicoTM 21, Life Technologies).
  • the obtained cell pellet was fixed and stained with primary antibodies solution containing anti-ki67 and anti-K5 antibodies.
  • the cells suspension was then incubated with the primary antibodies overnight at 4°C and thereafter, a secondary antibody solution containing goat anti-mouse Alexa Fluor 488 and goat anti-rabbit Alexa Fluor 594 antibodies were used to stain the cells.
  • the cell suspension was washed with PBS twice before analyzing in ArthurTM fluorescence cell counter.
  • both unstained kerCTs and K5 -stained kerCTs were used.
  • kerCTs cultured in 2D culture flasks at 80% confluence were trypsinized and the cell suspension was stained with primary antibody, anti-K5, followed by secondary antibody Alexa Fluor 488.
  • Both unstained and K5-antibody stained kerCTs were then analyzed under the ArthurTM fluorescence cell counter.
  • the cells were first gated for green fluorescent protein (GFP) and red fluorescent protein (RFP) signals based on the background signal from the unstained cells.
  • GFP green fluorescent protein
  • RFP red fluorescent protein
  • the GFP signals which correspond to K5 -expressing cells were then gated such that all the analyzed cells were included for quantification within the gating parameter, i.e., 100% of K5-positive cells. This is because all the cells analyzed should express K5 as they are all keratinocytes.
  • Cells digested from GelMA core-shell microspheres were then gated using the same parameters obtained. The percentage of proliferative kerCTs in monoculture and co-culture systems were then quantified based on Equation 5, where cells expressing both ki67 and K5 (indicating proliferative kerCTs) were normalized against the total number of cells expressing K5 (total number of kerCTs).
  • GelMA-DS40 and GelMA-DS90 macromers were dissolved in phosphate-buffered saline (PBS, Life Technologies) to achieve a stock concentration of 20% (w/v) GelMA-DS40 solution and 20% (w/v) GelMA-DS90 solution respectively.
  • a stock sodium alginate solution was also prepared by dissolving 2% (w/v) sodium alginate (NF, Spectrum Chemical Manufacturing Corp., New Brunswick, USA) in Milli-Q water. The stock solutions were then sterile-filtered through Minisart® polyethersulfone 0.22pm syringe filter (Sartorius, Gottingen, Germany).
  • HDFs and kerCTs were detached from culture flasks using 0.05% Trypsin-EDTA (Life Technologies) and the cells were collected by centrifuging at 1200 rpm for 5 minutes. The cell pellet collected was then re-suspended in PBS (Life Technologies). A fixed volume of cell suspension and 2% sodium alginate (for GelMA/alginate hydrogels) was then mixed with a varying volume of PBS and GelMA stock solutions to achieve a final GelMA concentration of 10% GelMA-DS40 with 4xl0 6 cells/mL HDFs and 10% GelMA - DS90/0.5% alginate with cell concentration of 20xl0 6 cells/mL kerCTs.
  • the core-shell solutions with two different cells were infused through a co-axial nozzle of 16-21G at a core-shell flow rate of 2.5: 12.5 mL/hr. Under an applied voltage of 9kV, the two solutions were sprayed into core-shell microspheres encapsulating HDFs and kerCTs in their respective core and shell compartment. The microspheres were collected in a lOOmM BaCh collector bath and UV-crosslinked under 365nm for 5 minutes at a fixed height.
  • the fully crosslinked cell-laden GelMA core-shell microspheres were then filtered through a 100pm cell strainer and washed thoroughly with a 150mM NaCl solution to remove any excess BaCh that may harm the cells (ionic displacement of Ba 2+ by Na + ).
  • the filtered cell-laden GelMA core-shell microspheres were then transferred to a 15 mL centrifuge tube by pipetting the skin co-culture media onto the cell strainer.
  • the cell-laden GelMA core-shell microspheres were resuspended in the co-culture media before aliquoting an equal volume for different cell studies.
  • An illustration of the experimental setup in fabricating the cell-laden GelMA core-shell microspheres is shown in Figure 5.
  • the microspheres were cultured in a chemically defined serum-free skin co-culture media (Atlantis Bioscience, Singapore). To ensure the volume of media accessed by the cells is uniform across the samples and minimize the loss of microspheres during media change, the cell-laden microspheres were cultured in 100pm cell strainers (Sigma).
  • Type A gelatin methacrylate (GelMA) was first fabricated via a facile one -pot synthesis between Type A gelatin and methacrylic anhydride (MAA) as described in the methodology section.
  • MAA methacrylic anhydride
  • the viscoelastic behavior of GelMA during the spraying process can be determined by investigating the effect of shear rate on the viscosity of GelMA solutions with varying DS at 25°C.
  • the GelMA concentration was fixed at 15% (w/v) across the different solutions.
  • GelMA-DS40 and DS90 solutions exhibited the largest difference in viscosity across the different shear rates, which results in a higher probability they do not mix during the spraying process. GelMA-DS40 and GelMA-DS90 are therefore selected as exemplary candidates for the core- shell solutions.
  • GelMA DS 90 hydrogel was found to exhibit a significantly higher storage modulus as compared to GelMA DS40, after 5 minutes of photocrosslinking.
  • GelMA-DS90 is thus selected as the shell solution for the fabrication of coreshell microspheres so that the stiffer, higher crosslinked shell can protect the softer GelMA - DS40 core after spraying and UV-crosslinked into microspheres.
  • 0.5% of sodium alginate was also added to the GelMA-DS90 shell solution to allow a quick crosslinking upon collection in a divalent cationic collector bath.
  • the cross-sectional SEM micrographs reveal the honeycomb structure of the GelMA-DS40 and DS90 hydrogels fabricated under different GelMA concentrations ( Figure 9 A).
  • the honeycomb structure is critical for the adhesion of cells while facilitating the diffusion of nutrients, oxygen, and waste exchange to the encapsulated cells.
  • GelMA concentration on mechanical properties of GelMA hydrogels [00197] GelMA concentration also has an effect on the stiffness of GelMA hydrogels. As shown in Figures 10A and 10B, the storage modulus of GelMA hydrogels increased with an increase in GelMA concentrations for both low and high GelMA-DS. The average storage modulus of GelMA hydrogels at the end of the 5 minutes of UV-crosslinking was quantified and presented in Figure 10C, which demonstrated that increasing GelMA concentration from 5%, 10% to 15% increases the storage modulus of hydrogels for both GelMA-DS. This is due to the increased availability of cross-linkable methacryloyl groups as GelMA concentration increases, resulting in higher crosslinking density as seen from the SEM micrographs in Figure 9A.
  • HDFs primary neonatal human dermal fibroblasts
  • kerCTs hTERT- immortalized primary neonatal human keratinocytes
  • HDFs suspension was then mixed with GelMA-DS40 solutions of different concentrations at a cell density of IxlO 5 cells/mL and the cell-laden GelMA prepolymer solutions were UV cross-linked for 5 minutes.
  • HDFs-laden GelMA hydrogels were then cultured over 7 days in their culture medium and the cell proliferation was quantified using CCK-8 assay.
  • the thickness of the shell layer plays an important role in the transfer of nutrients and oxygen from the external environment to the encapsulated skin cells. In circumstances where the shell layer is too thick, it will limit the diffusion of nutrients and waste exchange to the encapsulated cells, especially those in the core layer of the core-shell. This may result in a hypoxic core where the lack of oxygen will reduce the cell viability and capability to proliferate.
  • alginate a component of the shell material
  • FITC fluorescein isothiocyanate
  • the relationship between shell thickness and shell flow rate can be modeled, for example, according to a linear fit equation derived from the quantified data, allowing the estimation of the shell thickness in microspheres fabricated at any specific core-shell flow rate.
  • the shell thickness of microspheres should be thick enough to encapsulate the five-fold higher of keratinocytes relative to the fibroblasts but at the same time, the shell thickness should allow sufficient oxygen and nutrient exchange between the external environment and the encapsulated cells.
  • fabricating the core-shell microspheres at a core-shell flow rate of 1:14 mL/hr produced microspheres with large shell thickness, making up 93% of its total area. This shell thickness would be too thick for efficient diffusion and exchange of nutrients, oxygen and waste between the external environment and encapsulated cells, especially so for the fibroblasts encapsulated in the core of the microspheres.
  • fabricating at a core-shell flow rate of 5:10 mL/hr produced core-shell microspheres with very thin shell that makes up only 30% of its total area. A shell too thin will greatly limit the space for the high concentrated keratinocytes to grow in which may then compromise on the viability of the keratinocytes.
  • the core-shell flow rate of 2.5:12.5 mL/hr was found to be able to fabricate microspheres with an optimal shell thickness of 70% of its total area, a shell thick enough to encapsulate the high-density keratinocytes while having a relatively larger core compartment for oxygen and nutrient to diffuse more efficiently to the encapsulated fibroblasts.
  • co-axial electrospray system renders the capability to fine-tune the thickness of the shell layer just by controlling the core-shell flow rate and allows the encapsulation of two different skin cells in their desired co-culture ratio.
  • the optimal shell thickness could provide a biological environment that supports the growth of the encapsulated skin cells and provide for the recapitulation of the keratinocytes-fibroblasts crosstalk in the native skin.
  • HDFs The close proximity of HDFs with each other is required to promote autocrine signals essential for cell survival, as seen by the difference in growth between HDFs encapsulated in low density (3xl0 6 cells/mL) and high density (4xl0 6 cells/mL).
  • the GelMA-alginate core-shell microspheres were found to be fully digested by incubating the microspheres in 0.25% trypsin-EDTA for 5 minutes which is a typical cell dissociation method used in cell culture. This mild treatment with 0.25% trypsin-EDTA allows the GelMA-based microspheres to be readily degraded and release the cells without compromising on the cell viability or intracellular functions.
  • Trypsin is a type of serine protease that cleave peptide chains at the carboxyl side of positively charged amino acids such as lysine or arginine.
  • the specificity of trypsin in cleaving peptide chains of positively-charged amino acids is due to the presence of negatively-charged aspartate residue in the catalytic pocket (SI) of trypsin which attracts and stabilizes the positively charged amino acids.
  • GelMA contains cell-binding RGD motifs (Arg-Gly-Asp peptides) along with other free amine groups such as lysine and alanine, trypsin will cleave the peptide bonds of the positively charged arginine and lysine present in gelatin, allowing the digestion of GelMA microspheres.
  • trypsin-EDTA contains 380 mg/L of hydrated ethylenediaminetetraacetic acid (EDTA), which is a metal ion chelating agent. EDTA readily chelates with a metal ion and forms a stable bond between the nitrogen atom of EDTA and the metal ion.
  • the shell of GelMA core-shell microspheres consists of 0.5% alginate which upon contact with BaCh collection bath, crosslinks with barium ions to form barium alginate. The introduction of trypsin-EDTA during digestion thus chelates with the barium metal ions present in barium alginate, causing the crosslinked alginate to be dissolved.
  • Patient-derived cells of interest can be encapsulated into the relevant core or shell layer of the GelMA microspheres.
  • the GelMA microspheres can act like a ‘mini -bioreactor’, allowing the encapsulated cells to undergo cell expansion, and thereafter, before the GelMA microspheres are intentionally degraded with trypsin to release the cells for tissue regeneration purposes.
  • the high cell viability could also be attributed to the RGD motifs present in GelMA which facilitates cell adhesion upon encapsulation in the microgel.
  • the viability of the skin cells decreased to 44 ⁇ 0.57% (p ⁇ 0.05) and sustained at 40 ⁇ 9.84% after 7 days of culture.
  • the encapsulated skin cells exhibited high number of calcein-expressing live cells throughout the GelMA core-shell microspheres shown in Figure 16B, indicating that both the HDFs and kerCTs encapsulated in the core and shell compartments of the microspheres were highly viable.
  • the co-cultured cells were also observed to proliferate over 3 days as seen by the increase in number of Vybrant DiO (ThermoFisher) labelled MCF-7 cells in the core and CellTrackerTM CM-Dil (ThermoFisher) labelled E929 cells in the shell ( Figure 32).
  • Vybrant DiO ThermoFisher
  • CM-Dil CellTrackerTM CM-Dil
  • the aggregated cell cluster observed in co-cultured HDFs-kerCTs could be the interconnected network of HDFs surrounding the core of core-shell microspheres, which is confirmed by immunostaining kerCTs and HDFs as shown in Figure 22C.
  • the vimentin- expressing HDFs formed a small interconnected network in the core of the core-shell microspheres after 1 day of culture, and the cluster of HDF cells grew in size over the days. Following 7 days of culture, HDFs have elongated along the x-y and z- dimensions and proliferated to form a large 3D interconnected network that surrounds the core compartment of the core-shell microspheres.
  • Both HDFs and kerCTs were able to attach onto the well plate and proliferated to form a small patch of skin cells.
  • Vimentin-expressing HDFs were also successfully delivered from the core-shell microspheres seen in Figures 24G and 24H, albeit in lesser numbers than kerCTs. This is due to the encapsulation of kerCTs in the shell of the core-shell microspheres which allow the keratinocytes to be released first before HDFs could.
  • the capability to deliver both kerCTs and HDFs as well as the growth of the delivered skin cells into a large epidermal-dermal layer is critical in the re-epithelization of wounds; as the proliferative keratinocytes would help in the acceleration of wound closure while the dermal layer allows the recapitulation of functionality in the regenerated skin, providing structural integrity to the epithelial sheet, elasticity and a vascular bed.
  • core-shell solutions consisting of 5% GelMA-DS40 and 5% GelMA-DS90/0.5% alginate, respectively, were first electro- sprayed at different applied voltages shown in Figure 17.
  • the core-shell solutions were sprayed using 21-16G co-axial needle size and at a total flow rate of 15 mL/hr.
  • the fabricated microspheres were collected in a lOOmM BaC12 collector bath and UV-crosslinked for 5 minutes.
  • the concentration of shell solution was then increased to 10% GelMA-DS90/0.5% alginate and was found to form ovoid-shaped particles at 7.5kV and 9kV. While a better morphology of microspheres was observed when spayed at 10.5kV, the microspheres had a large variation in size ( Figure 18). A more monodispersed microspheres were achieved when sprayed with an optimal core-shell concentration of 10% GelMA-DS40 and 10% GelMA- DS90/0.5% alginate, at an applied voltage of 9kV.
  • the critical range of the electric field in which the stable cone -jet mode is formed is also dependent on the concentration of GelMA core- shell solutions. As the concentration of GelMA decreases, its viscosity decreases due to the lesser hindrance from the bulky MA side groups and hence lesser resistance to flow. As such, with the decrease in GelMA concentration, the lower viscosity and entanglement of GelMA polymer chains allows the GelMA molecular chains to move more freely, resulting in the deformation of the GelMA core-shell droplet at the tip of the nozzle before the voltage was applied.
  • the GelMA concentration also affects the stability of GelMA core-shell droplet at the nozzle tip which subsequently influences the critical range of electric field that can be applied to achieve a stable cone-jet mode.
  • Calcium chloride is a divalent ion that can crosslink with alginate for the fabrication of microspheres and is considered food-safe. It was therefore investigated as an alternative to BaCh as the collection bath. From Figure 19A, it was observed that microspheres collected in lOOmM CaCh were not able to form uniform spherical particles and had a wider size distribution as compared to those collected in BaCh. This is due to the lower ionic strength of Ca 2+ ions resulting in a weaker interaction between the cations and the negative charges in the alginate chains, and hence less stable core-shell microspheres.
  • the microspheres were fabricated at a core-shell flow rate of 2.5:12.5 mL/hr respectively and sprayed for a total of 3 minutes. 0.125ml of cells-laden core solution and 0.625 ml of cells- laden shell solution was tested. Thereafter, the microspheres were collected and transferred to a 15 mL centrifuge tube with phenol-red DMEM culture media and then to a Nunc® 90mm petri dish ( Figure 21A). The number of microspheres was quantified based on coverage.
  • co-axial electrospray thus brings forth several other advantages; the most notable advantage is that different cells can be encapsulated in the coreshell microspheres in a facile manner, as any additional procedures will reduce the viability of the encapsulated cells.
  • the independent flow of two different liquids prior to spraying means high molecular weight polymers can be used as long as its viscosity allows it to flow at working temperature and more critically, there is no surfactant involved in the fabrication of core-shell microspheres which if present, would be detrimental for the cells.
  • Other advantageous property of co-axial electrospray includes its scalability, reproducibility and ease of handling from the simple experimental set-up to the collection of core-shell microspheres. Unlike microfluidics which requires expertise and time to design, fabricate and optimise the device, co-axial electrospray proved to be a much simpler platform for the fabrication of scalable core-shell microspheres.
  • the present invention creates a highly versatile microsphere that encapsulates and co-delivers active ingredients such as cells, drugs or proteins in a facile, low-cost and high- yield manner, which renders it useful in a myriad of applications.
  • the microsphere When cells are encapsulated, the microsphere has a microenvironment that enables the encapsulated cells to retain their biological activity. As the microsphere has tunable properties, it would be possible to control the rate of release of active ingredients such as cells, drugs or proteins depending on the need of the subject.

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Abstract

The present invention relates to a microsphere with an inner core layer comprising a first cell type and an outer shell layer comprising a second cell type, and the use thereof. In an embodiment, the inner core comprises gelatin methacryloyl (GelMA); and the outer shell layer comprises gelatin methacryloyl (GelMA) and alginate. In another embodiment, the first cell type is fibroblasts and the second cell type is keratinocytes. The present invention also relates to the method of fabricating the microsphere.

Description

CORE-SHELL MICROSPHERES
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority of Singapore patent application No. 10202100488R, filed 15 January 2021, the contents of it being hereby incorporated by reference in its entirety for all purposes.
FIELD OF THE INVENTION
[0002] The present invention generally relates to biotechnology. In particular, the present invention relates to a microsphere with a core layer and a shell layer.
BACKGROUND OF THE INVENTION
[0003] Microspheres are known to be useful for the delivery of one or more active ingredients. Presently, microspheres are made by using synthetic polymers such as polylactic - co-glycolic acid (PLGA) and polylactic acid (PLLA), which allow for the delivery of small molecules for drug therapeutic applications. However, they have low biocompatibility for cell encapsulation, growth and delivery, therefore have limited use in cell-based and regenerative applications. In addition, the degradation rates of these microspheres are uncontrolled, therefore rendering it difficult for the user to control the rate of release of the active ingredients according to their needs.
[0004] Microspheres are presently fabricated by microfluidics using water-oil emulsions. However, the use of oil and surfactants limit their translation to the delivery of microspheres into human use. In addition, the yield of microspheres from microfluidics are relatively low and the fabrication method often requires multiple steps, which increases the time for fabrication. Furthermore, as highly skilled expertise is required in designing a microfluidic chip to cater to specific applications, microfluidics is limited in its versatility as a platform for diverse applications.
[0005] In view of the above problems, there is a need to provide an alternative microsphere composition that can co-deliver active ingredients in a controlled release manner and/or support cell growth. There is also a need to provide an alternative method of fabricating the micro sphere. SUMMARY OF THE INVENTION
[0006] In one aspect, there is provided a microsphere comprising: an inner core layer comprising gelatin methacryloyl (GelMA), wherein the inner core layer further comprises a first cell type; and an outer shell layer comprising gelatin methacryloyl (GelMA) and alginate, wherein the outer shell layer comprises a second cell type.
[0007] In another aspect, there is provided a synthetic skin replacement manufactured from the microsphere as disclosed herein.
[0008] In yet another aspect, there is provided a method of promoting wound healing comprising administering the microsphere as disclosed herein or a synthetic skin replacement as disclosed herein to a subject in need thereof.
[0009] In yet another aspect, there is provided a method of tissue regeneration comprising administering the microsphere as disclosed herein or a synthetic skin replacement as disclosed herein to a subject in need thereof.
[0010] In yet another aspect, there is provided a method of treating a disease comprising administering the microsphere as disclosed herein or a synthetic skin replacement as disclosed herein to a subject in need thereof.
[0011] In yet another aspect, there is provided a method of fabricating the microsphere as disclosed herein, the method comprising: a) preparing an inner core layer solution and an outer shell layer solution; b) setting a core flow rate and a shell flow rate, wherein the sum of the core flow rate and the shell flow rate results in a total flow rate of 9-19 ml/hr; c) electro spraying the microsphere.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:
[0013] Figure 1 shows exemplary core-shell microspheres and their uses. (A) A schematic 3D model of an exemplary core-shell microspheres using GelMA as core and GelMA and alginate as shell. The GelMA in the core can have low degree of substitution (DS) and the GelMA in the shell can have high degree of substitution. (B) Schematic 3D models of exemplary platforms of core-shell microspheres for encapsulation and co-delivery of (left) two different cell types for cell therapy or (right) two or more different drugs, proteins or nutrients for drug therapeutic application settings.
[0014] Figure 2 is a schematic illustration of methacrylate substitution with the primary amine of gelatin during gelatin methacryloyl (GelMA) synthesis.
[0015] Figure 3 is a photo of a cut planar section of fluorescein-labeled core- shell microspheres imaged under a confocal microscope. (Left) Fluorescence area of the shell, X, quantified using ImageJ; white outline demarcates the enclosed area of interest. (Right) Diameter of microsphere measured using ImageJ, as indicated by line Y.
[0016] Figure 4 is a schematic illustration of the experimental set-up of fabricating 3D GelMA core-shell microspheres through co-axial electrospray.
[0017] Figure 5 is a schematic illustration of an exemplary experimental set-up of fabricating 3D GelMA core-shell microspheres with HDFs (fibroblasts) and kerCTs (keratinocytes) encapsulated in their respective core-shell compartment through co-axial electro spraying. The same setup can be used for encapsulation of other cells as well as other non-cell components, such as different drugs, proteins or nutrients for different applications such as drug therapeutic applications or cell delivery in regenerative medicine applications.
[0018] Figure 6 is a 1 H-NMR spectra of pristine gelatin and fabricated GelMA with varying degrees of substitution. Peaks corresponding to (X) acrylic protons (2H) of lysine groups in methacrylamide grafts and those of hydroxyl lysine groups and (Y) methylene protons (2H) of unreacted lysine groups.
[0019] Figure 7 is a column graph that shows the average DS of GelMA samples fabricated under varying MAA/gelatin feed ratios. Error bars represent the standard deviation of means (n=3 batches). Figure 7 illustrates that increase in feed ratio of MAA to gelatin showed an increased degree of substitution.
[0020] Figure 8 is a line graph of the viscosity profile of 15% w/v GelMA solutions with different DS at 25°C. Data represents the average viscosity of n=3 GelMA solutions. Figure 7 illustrates that viscosity decreases with increasing shear rate.
[0021] Figure 9 illustrates the effects of GelMA concentrations and DS on pore size. (A) Representative cross-sectional SEM micrographs of freeze-dried (top panel) GelMA-DS40 and (bottom panel) GelMA DS 90 hydrogels fabricated under different GelMA concentrations. Scale bar = 100pm. (B) Column charts showing the pore size distribution of freeze-dried (left) GelMA-DS40 and (right) GelMA-DS90 hydrogels fabricated with different GelMA concentrations, obtained from n=50 pores.
[0022] Figure 10 illustrates the rheological analysis of the storage modulus of GelMA with different DS and different concentration. (A) A line graph representing the storage modulus of GelMA-DS40 with different concentrations (15%, 10%, 5%). (B) A line graph representing the storage modulus of GelMA-DS90 with different concentrations (15%, 10%, 5%). (C) A column graph representing the average storage modulus of 5%, 10% 15% w/v GelMA-DS40 and GelMA-DS90 after 5 minutes of UV-crosslinking. Data represents the average storage modulus of n=3 samples. Error bars represent the standard deviation of means. Comparison of means was done using one-way ANOVA with Tukey’s HSD post hoc test, *p<0.05, compared between the different GelMA groups. (D) A line graph representing the viscosity profile and (E) a line graph representing the storage moduli of 10% GelMA with different DS. (F) Representative brightfield images of core-shell microspheres fabricated with 10% GelMA of different DS as core material. Scalebar = 500pm.
[0023] Figure 11 illustrates the cell proliferation of cells in GelMA with different DS and different concentration. (A) A column graph showing the cell proliferation of HDFs in 5%, 10% and 15% (w/v) GelMA-DS40 hydrogels over 7 days. (B) A column graph showing the growth rate of HDFs (average increase of HDFs in cell number per day). Data represent proliferation/growth rate of HDFs in GelMA hydrogels (n=3) at each time point ± standard deviation of the mean. (C) A column graph showing the cell proliferation of kerCTs in 5%, 10% and 15% (w/v) GelMA-DS90/0.5% alginate hydrogels over 7 days. (D) A column graph showing the growth rate of kerCTs (average increase of kerCTs in cell number per day). Data represent proliferation/growth rate of kerCTs in GelMA/alginate hydrogels (n=3) at each time point ± standard deviation of the mean. Comparison of means was done using one-way ANOVA with Tukey’s HSD post hoc test, *p<0.05, compared between the different time points.
[0024] Figure 12 shows photos of representative cut planar section showing the FITC- conjugated shell and core of GelMA core-shell microspheres fabricated under core-shell flow rate of (A) 5:10 mE/hr (B) 2.5:12.5 mL/hr (C) 1:14 mL/hr respectively. Scalebar = 100pm.
[0025] Figure 13 is a line graph that showing the average % of shell thickness over cross- sectional area of core-shell microspheres fabricated under increasing shell flow rate (n=10 microspheres). [0026] Figure 14 are photos of representative brightfield images of mono-cultured HDFs cultured in the core of GelMA microspheres over 7 days. (Left to right) Microspheres sprayed using HDFs-laden GelMA solution with cell densities of IxlO6 cells/mL, 3xl06 cells/mL and 4xl06 cells/mL. Scalebar = 100pm.
[0027] Figure 15 is a column graph representing the cell viability of co-cultured kerCTs- HDFs in GelMA core-shell microspheres over 7 days. Data represent average cell viability of cell-laden microspheres cultured in three independent wells (n=3) at each time point ± standard deviation of the mean (viability). Comparison of means was done using one-way ANOVA with Tukey’s HSD post hoc test, *p<0.05, compared between the different time points.
[0028] Figure 16 shows the cell viability of co-cultured kerCTs-HDFs in GelMA core-shell microspheres over 7 days. (A) Representative fluorescent images of analysis of co-cultured skin cell viability in GelMA core-shell microspheres using confocal microscopy. Viable cells are presented in the left column, while dead cells are presented in the right column. Representative maximum intensity projection obtained from confocal stacks are presented. Scalebar = 100pm. (B) Representative fluorescent images of the distribution of live and dead cells in GelMA core-shell microspheres following 3 days of co-culture. Top panel is a gallery view of the z-stack images that show the distribution of live cells. Bottom panel is a gallery view of the z-stack images that show the distribution of dead cells. The number of dead cells is almost negligible. Gallery view of the obtained z-stack images are presented in steps of 20pm from the bottom to the top of the core-shell microspheres. Scalebar = 100pm. (C) Representative fluorescent images of the distribution of live and dead cells in GelMA coreshell microspheres following 5 days of co-culture. Top panel is a gallery view of the z-stack images that show the distribution of live cells. Bottom panel is a gallery view of the z-stack images that show the distribution of dead cells. Gallery view of the obtained z-stack images are presented in steps of 20pm from the bottom to the top of the core-shell microspheres. Scalebar = 100pm.
[0029] Figure 17 is a representative bright-field images of GelMA core-shell microspheres fabricated with different concentrations of core (GelMA-DS40) and shell (GelMA-DS90/0.5% alginate) solutions, under different applied voltage. Scale bar = 200pm.
[0030] Figure 18 provides column graphs representing the influence of different GelMA concentrations and applied voltage on the size distribution of fabricated core- shell microspheres, obtained from n=50 microspheres. [0031] Figure 19 illustrates size distribution of GelMA core-shell microspheres. (A) Representative brightfield images and size distribution of n=50 GelMA core-shell microspheres fabricated with different co-axial nozzle sizes and collected under different collector baths. Scale bar = 500pm. (B) Representative brightfield images and size distribution of n=50 GelMA core-shell microspheres fabricated under different total flow rates. Scale bar = 500pm.
[0032] Figure 20 provides (top) a representative brightfield image showing the morphology of the GelMA core-shell microspheres and (bottom) a column graph showing the size distribution of 200 randomly selected core-shell spheroids.
[0033] Figure 21 illustrates the yield and coverage analysis of the core-shell microspheres. (A) (left) A series of photos showing the collection of core-shell microspheres collected in DMEM culture media under 3 minutes of fabrication, (right) The same core-shell microspheres collected in DMEM culture media are displayed on a 9cm dish. (B) A representative image of a collage of the analysis of fluorescence area covered by FITC-conjugated core-shell microspheres using ImageJ software. Scale was set using the known diameter of the petri dish. The white outline demarcates the area analyzed by ImageJ.
[0034] Figure 22 illustrates the morphological characterization of cells in GelMA coreshell microspheres (A) Brightfield images of the morphological characterization of co-cultured skin cells in GelMA core-shell microspheres. (Left to right) Growth of co-cultured HDFs and kerCTs in the core and shell of microspheres (respectively) over 7 days as visualized at (top) 4x magnification and (bottom) lOx magnification. Brightfield imaging was used for day 0 to day 3 while phase contrast imaging was used to better visualize the morphology of released cells from degraded microspheres on day 7. Scalebar = 100pm. (B) Brightfield images of the morphological characterization of mono-cultured keratinocytes in GelMA core-shell microspheres. (Left to right) Growth of mono-cultured kerCTs in the shell of microspheres over 7 days as visualized at (top) 4x magnification and (bottom) lOx magnification. Brightfield imaging was used for day 0 to day 3 while phase contrast imaging was used to better visualize the morphology of released cells from degraded microspheres on day 7. Scalebar = 100pm. (C) Images of analysis of morphological growth of co-cultured HDFs-kerCTs in 3D GelMA core-shell microspheres by immunofluorescence and 3D reconstruction of confocal microscopy images. Cells were fixed and stained with antibodies against vimentin in HDFs, K5 in kerCTs and counter-stained with Hoechst. (I) Morphology of stained cells-laden microspheres presented as DIC image. Representative maximum intensity projection of BFP, GFP and RFP single channel corresponding to (II) Hoechst, (III) Vimentin and (IV) K5 staining respectively.
[0035] Figure 23 provides representative phase-contrast images of morphological characterization of delivered mono-cultured kerCTs at (A) 4x, (B) lOx and (C) 20x magnification following 7 days of culture in GelMA core-shell microspheres; and delivered co-cultured HDFs and kerCTs at (D) 4x, (E) lOx and (F) 20x magnification following 7 days of culture in GelMA core-shell microspheres. White arrow indicates kerCTs and the black arrow indicates HDFs as characterized based on morphological differences. Scale bar = 100pm. [0036] Figure 24 show representative images of delivered skin cells from GelMA coreshell microspheres after 7 days of co-culture. Cells were fixed and stained with antibodies against K5 in kerCTs (red) and vimentin in HDFs (green), counter- stained with Hoechst (blue). Representative phase-contrast images of degrading GelMA core-shell microspheres and delivered cells (A) before washing with PBS, and (B) after washing with PBS. Immunofluorescence staining of delivered skin cells under BFP, RFP and GFP single channel corresponding to Hoechst, K5 and Vimentin staining respectively at (C, E, G) 4x magnification and (D, F, H) at lOx magnification. Scalebar = 100pm.
[0037] Figure 25 provide representative images of immunofluorescence staining of growth of delivered kerCTs and encapsulated HDFs in 3D GelMA core-shell microspheres following 7 days of co-culture. Cells were fixed and stained with antibodies against K5 in kerCTs and vimentin in HDFs, counter-stained with Hoechst. (A) 4x and (B) lOx magnification of the merged immunofluorescence image. The single channel fluorescence images of (C) Hoechst, (D) K5 and (E) Vimentin staining. Scale bar = 100pm.
[0038] Figure 26 provides images of growth of delivered cells from 3D GelMA core-shell microspheres. (A) Representative brightfield images of growth of delivered kerCTs and HDFs from 3D GelMA core-shell microspheres following 14 days of co-culture. Scale bar = 100pm. (B) Representative brightfield images of growth of delivered kerCTs from 3D GelMA coreshell microspheres following 14 days of mono-culture. Scale bar = 100pm.
[0039] Figure 27 provides representative images of magnified fluorescence images depicting the propensity of HDFs to align and elongate along the sheet of kerCTs. Cells were fixed and stained with antibodies against K5 in kerCTs and vimentin in HDFs, counter- stained with Hoechst. The top panel (left to right) represents the phase contrast of delivered cells from GelMA core-shell microspheres before and after washing. A merged immunofluorescence image of all three channels was then presented. The bottom panel (left to right) represents the single channel fluorescence image of Hoechst, K5 and Vimentin staining. White line highlights the alignment of HDFs along the edge of kerCTs. Scale bar = 100pm.
[0040] Figure 28 provides representative images of fluorescence images depicting the proliferative state of delivered kerCTs and HDFs from 3D GelMA core-shell microspheres following 7 days of co-culture. Cells were fixed and stained with antibodies against ki67 (red) and vimentin in HDFs (green), counter-stained with Hoechst (blue). Phase contrast images of delivered cells from GelMA core-shell microspheres (A) before and (B) after washing. Immunofluorescence staining of delivered skin cells under BFP, GFP and RFP single channel corresponding to Hoechst, Vimentin and ki67 staining respectively at (C, F, I) 4x magnification, (D, G, J) at lOx magnification and (E, H, K) at 20x magnification. Scalebar = 100pm.
[0041] Figure 29 provides representative images of fluorescence images depicting the proliferative state of delivered kerCTs and HDFs from 3D GelMA core-shell microspheres following 14 days of co-culture. Cells were fixed and stained with antibodies against ki67 (red) and vimentin in HDFs (green), counter-stained with Hoechst (blue). Phase contrast images of delivered cells from GelMA core-shell microspheres (A) before and (B) after washing. Immunofluorescence staining of delivered skin cells under BFP, GFP and RFP single channel corresponding to Hoechst, Vimentin and ki67 staining respectively at (C, F, I) 4x magnification, (D, G, J) at lOx magnification and (E, H, K) at 20x magnification. Scalebar = 100pm.
[0042] Figure 30 is a column graph data representing average ki67-positive keratinocytes when mono-cultured and co-cultured within GelMA core-shell microspheres in three independent wells (n=3) at each time point ± SD. Comparison of means was done using oneway ANOVA with Tukey’s HSD post hoc test, *p<0.01 and *p<0.001, compared between cocultured kerCTs-HDFs and mono-cultured kerCTs at each time point. The # symbols indicate statistically significant differences (p<0.05) between a specific time point and the previous time point within the same group (e.g.: comparison between day 5 and day 3 of the mono-culture group). [0043] Figure 31 are representative brightfield images of co-cultured skin cells in GelMA microspheres before (left) and after digestion (right) using 0.25% Trypsin-EDTA. GelMA microspheres were fully digested and dissociated cells were released. Scalebar = 200pm.
[0044] Figure 32 provides representative images of immunofluorescence staining of growth of fluorescent labelled co-cultured cells (MCF7 cells in core layer and L929 cells in shell layer) over 3 days under different core-shell feed rate.
[0045] Figures 33A and B provide representative images of immunofluorescence staining captured from 2 different wells of a 6-well plate of kerCTs-HDFs viability after delivery from GelMA core-shell microspheres following 7 days of co-culture. Scalebar = 100pm.
DETAILED DESCRIPTION
[0046] Microspheres are small spherical particles that are commonly used as a delivery platform for active ingredients, for example, drugs, protein, cells, DNA or RNA. Microspheres can have 1 or more layers. For example, a core-shell microsphere has 2 layers, wherein the shell compartment conventionally provides protection of the encapsulated active ingredient against mechanical force during fabrication, enzymatic degradation, or host immune response. Presently, most core-shell microspheres are made by synthetic polymers, wherein their low biocompatibility renders them limited in their use for applications such as cell transplantation and regenerative medicine.
[0047] Recently, some studies have shown fabricated biocompatible core-shell microspheres that can encapsulate cells, however the core-shell microspheres are not able to stably support cell growth, and the rate of degradation of these microspheres are uncontrolled. This uncontrolled degradation means that the user is unable to control the rate of release of active ingredients or cells from the microsphere, therefore it would not be possible to tailor a microsphere with pre-programmed release profiles of active ingredients to match the needs of the user. Presently, there are no core-shell microspheres that can co-deliver active ingredients in a controlled release manner for different applications such as drug therapeutic applications or cell delivery in regenerative medicine applications.
[0048] In view of the above problems, the inventors have developed a microsphere that can support cell growth and/or have the ability to co-deliver cells and/or active ingredients in a controlled manner.
[0049] In one example, the microsphere of the present disclosure comprises: an inner core layer comprising gelatin methacryloyl (GelMA), wherein the inner core layer further comprises a first cell type; and an outer shell layer comprising gelatin methacryloyl (GelMA) and alginate, wherein the outer shell layer comprises a second cell type.
[0050] As used herein, the term “microsphere” refers to a small spherical particle with a diameter of 1-1000 pm. In one example, the microsphere has a diameter of, but is not limited to about 100-900 pm, about 200-800 pm, about 300-700 pm, about 400-600 pm, about SOO- SOO pm, or about 100 pm, about 200 pm, about 300 pm, about 400 pm, about 500 pm, about 600 pm, about 700 pm, about 800 pm, about 900 pm, or about 1000 pm. In another example, the microsphere has a diameter of about 356-408 pm. In another example, the microsphere has a diameter of about 382 pm. The microsphere can be fabricated using organic or inorganic materials, or a combination thereof. The microsphere can comprise an inner core layer and an outer shell layer, wherein the outer shell layer would encapsulate the inner core layer. As the outer shell layer confers protection to the inner core layer against physical elements such as mechanical force, or any biological elements such as enzymatic degradation or host immune response, in one example, the outer shell layer is stiffer than the inner core layer. The layers can be distinguished by the materials used to create the outer shell layer and the inner core layer, which would make it possible to determine the thickness of the outer shell layer.
[0051] As used herein, the terms “thickness of the outer shell layer” or “shell thickness” refer to the area of the shell and is determined by normalizing the fluorescence area of the outer shell (marked by X in Figure 3) against the planar cross-sectional area of the microsphere (7tr2) where r is the radius of the circle, and calculated using Equation 1 below:
S c'he Hll 4 t.h-ic ikness ( /n%/ \) = — Are -a of fluore -scenc-e x 100% Equat ..ion 1
Figure imgf000012_0001
[0052] The thickness of the shell is important for providing adequate space for the encapsulated cells to grow in the shell, and at the same time it must not be too thick as that could compromise the viability of the cells encapsulated in the core. The thickness of the shell is tunable depending on the applications of the microsphere. In one example, the thickness of the outer shell layer can be, but is not limited to about 5-99%, about 10-95%, about 20-95%, about 30-93%, about 40-80%, about 50-70% of the microsphere, or about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 93%, or about 95% of the microsphere. In one example, the thickness of the outer shell layer is about 70% of the microsphere.
[0053] Due to the tunable property of the micro sphere, the inner core layer and/or shell layer of the microsphere can be adjusted to comprise one or more active ingredients, for example, but not limited to cells, drugs, proteins, DNA or RNA. The choice of the active ingredient is dependent on the application that the microsphere is used for. For example, the microsphere can be fabricated to encapsulate cells in the core layer, in the shell layer, or in both the core and shell layers. The microsphere can further comprise one or more cell types. In one example, the inner core layer further comprises a first cell type. In another example, the outer shell layer further comprises a second cell type. In another example, the inner core layer further comprises a first cell type and the outer shell layer further comprises a second cell type. The first cell type in the inner core layer and the second cell type in the outer shell layer can be adjusted based on the requirements of the application in which the microsphere is used for. In one example, the first cell type and second cell type comprise anchorage dependent cells, non-anchorage dependent cells, or a combination thereof. As used herein, the term “anchorage dependent cells” refer to one or more cells that can grow, survive, or maintain function only when they are attached to a surface, such as extracellular matrix or tissue in a body, or glass or plastic when culturing the cells in vitro. Examples of such anchorage dependent cells include, but are not limited to, fibroblasts, keratinocytes, and stem cells such as mesenchymal stem cells. As used herein, the term “non-anchorage dependent cells” refer to one or more cells that can grow, survive, or maintain function when they are not attached to any surface. Examples of such nonanchorage dependent cells include, but are not limited to cancer stem cells, cancer cells, and hematopoietic cells such as bone marrow mononuclear cells or peripheral blood mononuclear cells.
[0054] The type of cells that are encapsulated in the core layer and shell layer of the microsphere are dependent on the application that the microsphere is used for. In one example, the inner core layer comprises a first cell type selected from a group consisting of fibroblasts, cancer cells, keratinocytes and stem cells. In another example, the first cell type is fibroblasts. In another example, the outer shell layer comprises a second cell type selected from a group consisting of keratinocytes, fibroblasts, epithelial cells, cancer cells, endothelial cells, and stem cells. In another example, the second cell type is keratinocytes. The microsphere can also comprise a combination of the first and second cell type as disclosed herein in the inner core layer and an outer shell layer respectively. In one example, the microsphere can further comprise an inner core layer comprising fibroblasts and an outer shell layer comprising keratinocytes. In another example, the microsphere can further comprise an inner core layer comprising cancer cells as described herein and an outer shell layer comprising fibroblasts. In another example, the microsphere can further comprise an inner core layer comprising breast cancer cells and an outer shell layer comprising fibroblasts.
[0055] Depending on the size of microsphere and the thickness of the outer shell layer, the cell density in the inner core layer and outer shell layer can also be adjusted. Vice versa, the size of microsphere and the thickness of the outer shell layer can be adjusted depending on the cell density in the inner core layer and outer shell layer. In one example, the cell density of the inner core layer is, but is not limited to about IxlO6 cells/ml to 6xl06 cells/ml, about 2xl06 cells/ml to 5xl06 cells/ml, about 3xl06 cells/ml to 4xl06 cells/ml, or about l.OxlO6 cells/ml, about 1.5xl06 cells/ml, about 2.0xl06 cells/ml, about 2.5xl06 cells/ml, about 3.0xl06 cells/ml, about 3.5xl06 cells/ml, about 4.0xl06 cells/ml, about 4.5xl06 cells/ml, about 5.0xl06 cells/ml, about 5.5xl06 cells/ml, or about 6.0xl06 cells/ml. In another example, the inner core layer comprises IxlO6 cells/ml to 4xl06 cells/ml fibroblasts. In another example, the inner core layer comprises 4xl06 cells/ml fibroblasts.
[0056] In one example, the cell density of the outer shell layer is, but is not limited to about 5xl06 cells/ml to 30xl06 cells/ml, about 10xl06 cells/ml to 25xl06 cells/ml, about 15xl06 cells/ml to 20xl06 cells/ml, or about 5.0xl06 cells/ml, about 6.0xl06 cells/ml, about 7.0xl06 cells/ml, about 8.0xl06 cells/ml, about 9.0xl06 cells/ml, about lO.OxlO6 cells/ml, about l l.OxlO6 cells/ml, about 12.0xl06 cells/ml, about 13.0xl06 cells/ml, about 14.0xl06 cells/ml, about 15.0xl06 cells/ml, about 16.0xl06 cells/ml, about 17.0xl06 cells/ml, about 18.0xl06 cells/ml, about 19.0xl06 cells/ml, about 20.0xl06 cells/ml, about 21.0xl06 cells/ml, about 22.0xl06 cells/ml, about 23.0xl06 cells/ml, about 24.0xl06 cells/ml, about 25.0xl06 cells/ml, about 26.0xl06 cells/ml, about 27.0xl06 cells/ml, about 28.0xl06 cells/ml, about 29.0xl06 cells/ml, or about 30.0xl06 cells/ml. In another example, the outer shell layer comprises 5xl06 cells/ml to 20xl06 cells/ml keratinocytes. In another example, the outer shell layer comprises 20xl06 cells/ml keratinocytes.
[0057] Gelatin methacryloyl (GelMA) is modified from natural polymer gelatin and retains the tri-amino acid sequence arginine-glycine-aspartic acid (RGD) sequences that gelatin contains to promote cell adhesion. GelMA has a porous micro structure, which provides an optimal environment for encapsulated cells to grow in as it allows diffusion of nutrients oxygen and waste exchange between the culture medium and encapsulated cells. GelMA is also biodegradable, therefore it is used as a material for both the inner core and the outer shell of the microsphere. The biodegradable feature of GelMA provides a time-controlled release of, for example, cells, small molecules or drugs. The time of degradation in either the core or shell layer of the microsphere and the release of, for example, cells can be controlled by adjusting the different parameters of GelMA, for example, the degree of methacryloyl substitution (DS) of GelMA.
[0058] As used herein, the terms “degree of substitution”, “degree of methacryloyl substitution” or “DS” refer to the number of substituent groups attached per base unit, in this case, the number of available cross-linkable methacryloyl groups (substituent group) that can crosslink with a photoinitiator crosslinker (base unit), for example, Lithium Phenyl (2,4,6- Trimethylbenzoyl) Phosphinate (LAP) or Irgacure. As used herein, the term “photoinitiator” refers to a compound that creates reactive species, for example, but not limited to, free radicals, cations or anions when exposed to radiation such as ultraviolet. Photoinitiators are used to initiate a crosslinking or polymerization process upon exposure to radiation. A higher degree of methacryloyation substitution indicates that there is more available cross -linkable methacryloyl groups to crosslink with the photoinitiator crosslinker. This results in a higher crosslinking density after UV irradiation and hence a stiffer hydrogel, which allows it to degrade slower. The degree of methacryloyl substitution is quantified by the 2,4,6- Trinitrobenzenesulfonic acid (TNBSA) method, and calculated using Equation 2 below:
, A (free amine groups of GelMA) >
GelMA DS % = 1 - — - - - — - - - x 100% Equation 2
A (free amine groups of gelatin)
[0059] In one example, the GelMA of the inner core layer comprises a degree of methacryloyl substitution (DS) that can be, but is not limited to about 35%-60%, about 35%- 55%, about 35%-50%, about 40%-55%, about 45%-50%, in particular about 38%-46%. In another example, the GelMA of the inner core layer comprises a degree of methacryloyl substitution (DS) that can be, but is not limited to about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, or about 60%. In another example, the DS of the GelMA of the inner core layer is about 41.73%.
[0060] In one example, the GelMA of the outer shell layer comprises a degree of methacryloyl substitution (DS) that can be, but is not limited to about 65%-96%, about 75%- 95%, about 85%-95%, in particular about 86%-95%. In another example, the GelMA of the outer shell layer comprises a degree of methacryloyl substitution (DS) that can be, but is not limited to about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,. In another example, the DS of the GelMA of the outer shell layer is about 90.74%. [0061] The different DS can be rounded to their nearest tens for ease of terminology. For example, the DS of GelMA that is 41.73%, 57.10% and 90.74% can be labeled as GelMA- DS40, GelMA-DS60 and GelMA-DS90 respectively. GelMA-DS90 is stiffer than the GelMA - DS40. Stiffness can be measured by the storage modulus. As used herein, the term “storage modulus” refers to the measure of the amount of energy required to be put into a material in order to distort it. Storage modulus provides an indication of the stiffness of a solid material. A higher storage modulus indicates a stiffer material. In one example, the inner core layer comprises a storage modulus of 1.30 Pa to 4.61 kPa. In another example, the inner core layer comprises a storage modulus that can be, but is not limited to about 50-500 Pa, about 500 Pa- 1 kPa, about 1-1.5 kPa, about 1.5-2 kPa, about 2-2.5 kPa, about 2.5-3 kPa, about 3-3.5 kPa, about 3.5-4 kPa, about 4-4.5 kPa, or about 500 Pa, about 1 kPa, about 1.5 kPa, about 2 kPa, about 2.5 kPa, about 3 kPa, about 3.5 kPa, about 4 kPa, or about 4.5 kPa. In another example, the inner core layer has a storage modulus of about 4.61 kPa. In another example, the inner core layer has a storage modulus of about 821 Pa.
[0062] For the outer shell layer to be stiffer than the inner core layer, the outer shell layer should have a higher storage modulus. In one example, the outer shell layer comprises a storage modulus of 14.96 Pa to 13.3 kPa. In another example, the inner core layer comprises a storage modulus that can be, but is not limited to about 50-500 Pa, about 500 Pa-1 kPa, about 1-1.5 kPa, about 1.5-2 kPa, about 2-2.5 kPa, about 2.5-3 kPa, about 3-3.5 kPa, about 3.5-4 kPa, about 4-4.5 kPa, about 4.5-5 kPa, about 5-5.5 kPa, about 5.5-6 kPa, about 6-6.5 kPa, about 6.5-7 kPa, about 7-7.5 kPa, about 7.5-8 kPa, about 8-8.5 kPa, about 8.5-9 kPa, about 9-9.5 kPa, about 9.5- 10 kPa, about 10-10.5 kPa, about 10.5-11 kPa, about 11-11.5 kPa, about 11.5-12 kPa, about 12-12.5 kPa, about 12.5-13 kPa, or about 500 Pa, about 1 kPa, about 1.5 kPa, about 2 kPa, about 2.5 kPa, about 3 kPa, about 3.5 kPa, about 4 kPa, about 4.5 kPa, about 5 kPa, about 5.5 kPa, about 6 kPa, about 6.5 kPa, about 7 kPa, about 7.5 kPa, about 8 kPa, about 8.5 kPa, about 9 kPa, about 9.5 kPa, about 10 kPa, about 10.5 kPa, about 11 kPa, about 11.5 kPa, about 12 kPa, about 12.5 kPa, or about 13 kPa. In another example, the outer shell layer has a storage modulus of about 13.3 kPa. In another example, the outer shell layer has a storage modulus of about 2.36 kPa.
[0063] In addition to adjusting the degree of methacryloyl substitution (DS) of GelMA, the time of degradation and the release of, for example, cells can also be controlled by adjusting the concentration of GelMA. In one example, the inner core layer and outer shell layer each comprise a concentration of about 5-15% (w/v) GelMA. In another example, the inner core layer and outer shell layer each comprise a concentration of, but is not limited to about 5% (w/v), about 6% (w/v), about 7% (w/v), about 8% (w/v), about 9% (w/v), about 10% (w/v), about 11% (w/v), about 12% (w/v), about 13% (w/v), about 14% (w/v), or about 15% (w/v). In a preferred example, the inner core layer and outer shell layer each have a concentration of about 10% (w/v) GelMA.
[0064] In addition to gelatin methacryloyl (GelMA), the outer shell layer of the micro sphere further comprises alginate. The addition and concentration of alginate in GelMA can also control the time of degradation and the release of, for example, cells. In one example, the outer shell layer comprises about 0.1- 1.0% alginate. In another example, the outer shell layer comprises about 0.5% alginate.
[0065] The combination of the degree of methacryloyl substitution (DS) and concentration of GelMA can affect the pore size of the inner core layer and outer shell layer of the microsphere. The pores in the inner core layer and outer shell layer of the microsphere influence the adhesion of cells while facilitating the diffusion of nutrients, oxygen, and waste exchange to the encapsulated cells. Having pore size that is too small can reduce the efficiency of diffusion of nutrients, oxygen, and waste exchange to the encapsulated cells. On the other hand, if the pore size is too large, the cells might not be able to adhere properly. In one example, the inner core layer comprises a pore size of, but is not limited to, about 10 - 70 pm, about 20 - 60 pm, about 30 - 50 pm, about 35 - 45 pm, or about 20 pm, about 21 pm, about 22 pm, about 23 pm, about 24 pm, about 25 pm, about 26 pm, about 27 pm, about 28 pm, about 29 pm, about 30 pm, about 31 pm, about 32 jam, about 33 |am, about 34 pm, about 35 |am, about 36 |am, about 37 pm, about 38 |am, about 39 |am, about 40 |am, about 41 pm, about 42 pm, about 43 pm, about 44 pm, about 45 |am, about 46 |am, about 47 pm, about 48 |am, about 49 |am, about 50 |am, about 51 |am, about 52 pm, about 53 |am, about 54 pm, about 55 |am, about 56 |am, about 57 pm, about 58 |am, about 59 |am, about 60 |am, about 61 |am, about 62 jam, about 63 pm, about 64 pm, about 65 |am, about 66 |am, about 67 pm, about 68 |am, about 69 |am or about 70 |am. In another example, the inner core layer comprises a pore size of about 13 - 65 pm. In another example, the inner core layer pore size is about 24.05 pm.
[0066] In one example, the outer shell layer comprises a pore size of, but is not limited to, about 20 - 50 pm, about 25 - 45 pm, about 30 - 40 pm, or about 20 pm, about 21 pm, about 22 pm, about 23 pm, about 24 pm, about 25 pm, about 26 pm, about 27 pm, about 28 pm, about 29 pm, about 30 pm, about 31 pm, about 32 pm, about 33 pm, about 34 pm, about 35 pm, about 36 pm, about 37 pm, about 38 pm, about 39 pm, about 40 pm, about 41 pm, about 42 pm, about 43 pm, about 44 pm, about 45 pm, about 46 pm, about 47 pm, about 48 pm, about 49 pm, or about 50 pm. In another example, the outer shell layer comprises a pore size of about 27 - 39 pm. In another example, the outer shell layer pore size is about 36.83 pm.
[0067] It can be appreciated that the microsphere as disclosed herein is tunable by combining different parameters as disclosed herein to meet the requirements of the application in which the microsphere is used for. The microsphere can be cell-free. In one example, the microsphere comprises: an inner core layer comprising 5-15% (w/v) gelatin methacryloyl (GelMA) and a storage modulus of 1.30 Pa to 4.61 kPa; an outer shell layer comprising 5-15% (w/v) gelatin methacryloyl (GelMA), a storage modulus of 14.96 Pa to 13.3 kPa, and alginate; wherein the thickness of the outer shell layer is about 5-99% of the microsphere. In another example, the microsphere comprises: an inner core layer comprising 5-15% (w/v) gelatin methacryloyl (GelMA) and a pore size of 13-65 pM; an outer shell layer comprising 5-15% (w/v) gelatin methacryloyl (GelMA), a pore size of 27-39 pM, and alginate; wherein the thickness of the outer shell layer is about 5-99% of the microsphere. In another example, the microsphere comprises: an inner core layer comprising 5-15% (w/v) gelatin methacryloyl (GelMA) with a degree of methacryloyl substitution (DS) of about 30-60%; an outer shell layer comprising 5-15% (w/v) gelatin methacryloyl (GelMA) with a degree of methacryloyl substitution (DS) of about 65-96% and alginate; wherein the thickness of the outer shell layer is about 5-99% of the microsphere. [0068] The microspheres as disclosed herein can encapsulate one or more types of cells in the core and/or shell layer. In one example, the microsphere comprises: an inner core layer comprising 5-15% (w/v) gelatin methacryloyl (GelMA), a storage modulus of 1.30 Pa to 4.61 kPa, and a first cell type; an outer shell layer comprising 5-15% (w/v) gelatin methacryloyl (GelMA), a storage modulus of 14.96 Pa to 13.3 kPa, and alginate; wherein the thickness of the outer shell layer is about 5-99% of the microsphere. In another example, the microsphere comprises: an inner core layer comprising 5-15% (w/v) gelatin methacryloyl (GelMA), a pore size of 13-65 pM, and a first cell type; an outer shell layer comprising 5-15% (w/v) gelatin methacryloyl (GelMA), a pore size of 27-39 pM, and alginate; wherein the thickness of the outer shell layer is about 5-99% of the microsphere. In another example, the microsphere comprises: an inner core layer comprising 5-15% (w/v) gelatin methacryloyl (GelMA) with a degree of methacryloyl substitution (DS) of about 30-60% and and a first cell type; an outer shell layer comprising 5-15% (w/v) gelatin methacryloyl (GelMA) with a degree of methacryloyl substitution (DS) of about 65-96% and alginate; wherein the thickness of the outer shell layer is about 5-99% of the microsphere. In another example, the microsphere comprises: an inner core layer comprising 5-15% (w/v) gelatin methacryloyl (GelMA) and a storage modulus of 1.30 Pa to 4.61 kPa; an outer shell layer comprising 5-15% (w/v) gelatin methacryloyl (GelMA), a storage modulus of 14.96 Pa to 13.3 kPa, alginate and a second cell type; wherein the thickness of the outer shell layer is about 5-99% of the microsphere. In another example, the microsphere comprises: an inner core layer comprising 5-15% (w/v) gelatin methacryloyl (GelMA) and a pore size of 13-65 pM; an outer shell layer comprising 5- 15% (w/v) gelatin methacryloyl (GelMA), a pore size of 27-39 pM, alginate, and a second cell type; wherein the thickness of the outer shell layer is about 5-99% of the microsphere. In another example, the microsphere comprises: an inner core layer comprising 5-15% (w/v) gelatin methacryloyl (GelMA) with a degree of methacryloyl substitution (DS) of about 30- 60%; an outer shell layer comprising 5-15% (w/v) gelatin methacryloyl (GelMA) with a degree of methacryloyl substitution (DS) of about 65-96%, alginate, and a second cell type; wherein the thickness of the outer shell layer is about 5-99% of the microsphere.
[0069] In one example, the microsphere comprises: an inner core layer comprising 5-15% (w/v) gelatin methacryloyl (GelMA), a storage modulus of 1.30 Pa to 4.61 kPa and a first cell type; an outer shell layer comprising 5-15% (w/v) gelatin methacryloyl (GelMA), a storage modulus of 14.96 kPa to 13.3 kPa, alginate and a second cell type; wherein the thickness of the outer shell layer is about 5-99% of the microsphere. In another example, the microsphere comprises: an inner core layer comprising 5-15% (w/v) gelatin methacryloyl (GelMA), a pore size of 13-65 pM and a first cell type; an outer shell layer comprising 5-15% (w/v) gelatin methacryloyl (GelMA), a pore size of 27-39 pM, alginate and a second cell type; wherein the thickness of the outer shell layer is about 5-99% of the microsphere. In another example, the microsphere comprises: an inner core layer comprising 5-15% (w/v) gelatin methacryloyl (GelMA) with a degree of methacryloyl substitution (DS) of about 30-60% and a first cell type; and an outer shell layer comprising 5-15% (w/v) gelatin methacryloyl (GelMA) a degree of methacryloyl substitution (DS) of about 65-96%, alginate, wherein the thickness of the outer shell layer is about 5-99% of the microsphere. In another example, the microsphere comprises: an inner core layer comprising 5-15% (w/v) gelatin methacryloyl (GelMA) with a degree of methacryloyl substitution (DS) of about 30-60% and a first cell type; and an outer shell layer comprising 5-15% (w/v) gelatin methacryloyl (GelMA) a degree of methacryloyl substitution (DS) of about 65-96%, 0.1-1.0% alginate, and a second cell type, wherein the thickness of the outer shell layer is about 5-99% of the microsphere. In another example, the microsphere comprises: an inner core layer comprising 10% (w/v) gelatin methacryloyl (GelMA) with a degree of methacryloyl substitution (DS) of about 41.73% and a first cell type; and an outer shell layer comprising 10% (w/v) gelatin methacryloyl (GelMA) a degree of methacryloyl substitution (DS) of about 90.74%, 0.5% alginate, and a second cell type, wherein the thickness of the outer shell layer is about 70% of the microsphere. In another example, the microsphere comprises: an inner core layer comprising 10% (w/v) gelatin methacryloyl (GelMA) with a degree of methacryloyl substitution (DS) of about 41.73% and fibroblasts; and an outer shell layer comprising 10% (w/v) gelatin methacryloyl (GelMA) a degree of methacryloyl substitution (DS) of about 90.74%, 0.5% alginate, and keratinocytes, wherein the thickness of the outer shell layer is about 70% of the microsphere.
[0070] The microspheres as disclosed herein have tunable properties for different applications by fine-tuning the different hydrogel formulations that are used for both core and shell compartment. By tailoring the formulations of the inner core and outer shell layers of the microsphere as described herein, different mechanical strength and degradation rates can be achieved, resulting in a tunable diffusion and release time of co-encapsulated active ingredients or cells according to the needs of applications. The application can be, but is not limited to manufacturing therapeutic products, delivery of a 2D or 3D co-cultured cell in a biomedical setting, therapeutic drug or small molecules delivery, and food science. In one example, there is a composition that comprises the microsphere as disclosed herein. In another example, there is provided a synthetic skin replacement that is manufactured from the microsphere as disclosed herein. Another example of the application is the method of manufacturing a synthetic skin replacement using the microsphere as disclosed herein. In one example, the synthetic skin replacement is selected from a group consisting of an epidermal-dermal skin sheet, an epidermal skin sheet and a dermal skin sheet.
[0071] It is envisaged that the microsphere, synthetic skin replacement or composition as disclosed herein can be used in a clinical setting and be used to treat a subject in need thereof. In one example, there is provided a method of promoting wound healing comprising administering the microsphere as disclosed herein or a synthetic skin replacement as disclosed herein to a subject in need thereof. In another example, the microsphere as disclosed herein or a synthetic skin replacement as disclosed herein is for use in promoting wound healing. In another example, there is provided the use of the microsphere as disclosed herein or a synthetic skin replacement as disclosed herein in the manufacture of a medicament for promoting wound healing. In another example, there is provided a method of tissue regeneration comprising administering the microsphere as disclosed herein or a synthetic skin replacement as disclosed herein to a subject in need thereof. In another example, the microsphere as disclosed herein or a synthetic skin replacement as disclosed herein is for use in tissue regeneration. In another example, there is provided the use of the microsphere as disclosed herein or a synthetic skin replacement as disclosed herein in the manufacture of a medicament for tissue regeneration. In another example, there is provided a method of treating a disease comprising administering the microsphere as disclosed herein or a synthetic skin replacement as disclosed herein in a subject in need thereof. In another example, the microsphere as disclosed herein or a synthetic skin replacement as disclosed herein is for use in treating a disease. In another example, there is provided the use of the microsphere as disclosed herein or a synthetic skin replacement as disclosed herein in the manufacture of a medicament for treating a disease. The disease can be a skin disease or disorder, wherein the skin disease or disorder comprises bum injury, Recessive Dystrophic Epidermolysis Bullosa, diabetic foot ulcers, infectious wounds, ischemic wounds, open wounds and chronic wound. In one example, the administration comprises topical, subcutaneous, intravenous, or intramuscular administration. [0072] Prior to treating a subject in need thereof, the method can further comprise pretreating the microsphere as disclosed herein with trypsin before administration. The trypsin can be any trypsin that is commonly used, for example, 0.25% trypsin EDTA. The microsphere can be pre-treated for at least 5 minutes or for about 5 minutes.
[0073] In order to be able to produce the microspheres on a large-scale, it is pertinent that the method of fabricating the microsphere has high tunability, high yield, high scalability and high encapsulation efficiency. As used herein, the term “yield” refers to the percentage of nondefective items of all produced items, as indicated by the ratio of the number of non-defective items against the number of manufactured items. Ideally, 100% of all the materials used can be directly sprayed into microspheres, unlike in other fabrication process where some of the materials are lost due to external environment (heat, stirring), or through chemical reaction means. As disclosed herein, the present invention discloses a method of fabricating the microsphere that can have 100% yield microspheres, as well as the fabrication of a large amount of microspheres in a short amount of time.
[0074] The method of fabricating the microsphere as disclosed herein comprises: a) preparing an inner core layer solution and an outer shell layer solution; b) setting a core flow rate and a shell flow rate, wherein the sum of the core flow rate and the shell flow rate results in a total flow rate of 9-19 ml/hr; c) electro spraying the microsphere.
[0075] The shell thickness of the core-shell microspheres can be facilely tuned by changing the flow rate of core and shell hydrogel solutions, thereby tuning the release of encapsulated core ingredients. In one example, the total flow rate of 15 ml/hr.
[0076] When the total flow rate is 15 ml/hr, in one example, the core flow rate is about 1- 14 ml/hr, and it would be apparent that the shell flow rate is about 14-1 ml/hr. In another example, the following combination can be, but is not limited to: the core flow rate is about 1 ml/hr and the shell flow rate is about 14 ml/hr, the core flow rate is about 2 ml/hr and the shell flow rate is about 13 ml/hr, the core flow rate is about 3 ml/hr and the shell flow rate is about 12 ml/hr, the core flow rate is about 4 ml/hr and the shell flow rate is about 11 ml/hr, the core flow rate is about 5 ml/hr and the shell flow rate is about 10 ml/hr, the core flow rate is about 6 ml/hr and the shell flow rate is about 9 ml/hr, the core flow rate is about 7 ml/hr and the shell flow rate is about 8 ml/hr, the core flow rate is about 8 ml/hr and the shell flow rate is about 7 ml/hr, the core flow rate is about 9 ml/hr and the shell flow rate is about 6 ml/hr, the core flow rate is about 10 ml/hr and the shell flow rate is about 5 ml/hr, the core flow rate is about 11 ml/hr and the shell flow rate is about 4 ml/hr, the core flow rate is about 12 ml/hr and the shell flow rate is about 3 ml/hr, the core flow rate is about 13 ml/hr and the shell flow rate is about 2 ml/hr, or the core flow rate is about 14 ml/hr and the shell flow rate is about 1 ml/hr. In another example, the core flow rate is about 2.5 ml/hr and the shell flow rate is about 12.5ml/hr. [0077] The inner core layer solution comprises gelatin methacryloyl (GelMA) as disclosed herein. In one example, the GelMA of the inner core layer solution comprises a degree of methacryloyl substitution (DS) of about 35%-60%. In another example, the DS of the GelMA of the inner core layer solution is about 41.73%.
[0078] The outer shell layer solution comprises gelatin methacryloyl (GelMA) and alginate as disclosed herein. In one example, the GelMA of the outer shell layer solution comprises a degree of methacryloyl substitution (DS) of about 65%-96%. In another example, the DS of the GelMA of the outer shell layer solution is about 90.74%.
[0079] In one example, the outer shell layer solution comprises about 0.1- 1.0% alginate. In another example, the outer shell layer solution comprises about 0.5% alginate.
[0080] The inner core layer solution and outer shell layer solution each further comprise a photoinitiator. In one example, the photoinitiator comprises Lithium Phenyl (2,4,6- Trimethylbenzoyl) Phosphinate (LAP) or Irgacure. In another example, the photoinitiator is Lithium Phenyl (2,4,6-Trimethylbenzoyl) Phosphinate (LAP). In another example, the photoinitiator is 0.1 % LAP.
[0081] In one example, the inner core layer solution and outer shell layer solution each comprise a concentration of about 5-15% (w/v) GelMA. In another example, the inner core layer and outer shell layer each comprise a concentration of, but is not limited to about 5% (w/v), about 6% (w/v), about 7% (w/v), about 8% (w/v), about 9% (w/v), about 10% (w/v), about 11% (w/v), about 12% (w/v), about 13% (w/v), about 14% (w/v), or about 15% (w/v). In a preferred example, the inner core layer and outer shell layer each have a concentration of about 10% (w/v) GelMA.
[0082] In one example, the inner core layer solution can further comprise a first cell type. In another example, the outer shell solution can further comprise a second cell type. The first cell type in the inner core layer solution and the second cell type in the outer shell layer solution can be adjusted based on the requirements of the application in which the microsphere is used for. In one example, the first cell type and second cell type comprise anchorage dependent cells as disclosed herein, non-anchorage dependent cells as disclosed herein, or a combination thereof. In one example, the first cell type is selected from a group consisting of fibroblasts, cancer cells, keratinocytes and stem cells. In another example, the first cell type is fibroblasts. In another example, the second cell type is selected from a group consisting of keratinocytes, fibroblasts, epithelial cells, cancer cells, endothelial cells, and stem cells. In another example, the second cell type is keratinocytes.
[0083] The cell density of in the inner core layer solution and outer shell layer solution can also be adjusted. In one example, the cell density of the inner core layer solution is, but is not limited to about IxlO6 cells/ml to 6xl06 cells/ml, about 2xl06 cells/ml to 5xl06 cells/ml, about 3xl06 cells/ml to 4xl06 cells/ml, or about l.OxlO6 cells/ml, about 1.5xl06 cells/ml, about 2.0xl06 cells/ml, about 2.5xl06 cells/ml, about 3.0xl06 cells/ml, about 3.5xl06 cells/ml, about 4.0xl06 cells/ml, about 4.5xl06 cells/ml, about 5.0xl06 cells/ml, about 5.5xl06 cells/ml, or about 6.0xl06 cells/ml. In another example, the inner core layer solution comprises a cell density of IxlO6 cells/ml to 4xl06 cells/ml fibroblasts. In another example, the inner core layer solution comprises a cell density of 4xl06 cells/ml fibroblasts.
[0084] In one example, the cell density of the outer shell layer solution is, but is not limited to about 5xl06 cells/ml to 30xl06 cells/ml, about 10xl06 cells/ml to 25xl06 cells/ml, about 15xl06 cells/ml to 20xl06 cells/ml, or about 5.0xl06 cells/ml, about 6.0xl06 cells/ml, about 7.0xl06 cells/ml, about 8.0xl06 cells/ml, about 9.0xl06 cells/ml, about lO.OxlO6 cells/ml, about l l.OxlO6 cells/ml, about 12.0xl06 cells/ml, about 13.0xl06 cells/ml, about 14.0xl06 cells/ml, about 15.0xl06 cells/ml, about 16.0xl06 cells/ml, about 17.0xl06 cells/ml, about 18.0xl06 cells/ml, about 19.0xl06 cells/ml, about 20.0xl06 cells/ml, about 21.0xl06 cells/ml, about 22.0xl06 cells/ml, about 23.0xl06 cells/ml, about 24.0xl06 cells/ml, about 25.0xl06 cells/ml, about 26.0xl06 cells/ml, about 27.0xl06 cells/ml, about 28.0xl06 cells/ml, about 29.0xl06 cells/ml, or about 30.0xl06 cells/ml. In another example, the outer shell layer solution comprises a cell density of 5xl06 cells/ml to 20xl06 cells/ml keratinocytes. In another example, the outer shell layer solution comprises a cell density of 20xl06 cells/ml keratinocytes.
[0085] Step c) of the method of fabricating the microsphere as disclosed herein can comprise a co-axial nozzle for electro spraying. In one example, the co-axial nozzle is 16-21G or 18-14G.
[0086] During step c), an applied voltage needs to be set for electro spraying. In one example, the applied voltage can be, but is not limited to, about 7.5 - 12kV, about 8 - l lkV, about 9 - lOkV, or about 7.5kV, about 8.0kV, about 8.5kV, about 9.0kV, about 9.5kV, about lO.OkV, about 10.5kV, or about 1 l.OkV. In another example, the applied voltage is about 9kV. [0087] The method of fabricating the microsphere further comprises d) collecting the microsphere in BaCh or CaCh. In one example, the concentration of BaCh or CaCh can be about 50mM to about 150mM. In another example, the concentration of BaCh or CaCh can be, but is not limited to about 50mM, about 60mM, about 70mM, about 80mM, about 90mM, about lOOmM, about l lOmM, about 120mM, about 130mM, about 140mM or about 150mM. In another example, the concentration of BaCh or CaCh is lOOmM.
[0088] In one example, the method of fabricating the microsphere further comprises e) exposing the microsphere from step d) to ultraviolet.
[0089] Figure 4 provides an exemplary method as disclosed herein, wherein the method of fabricating the microsphere comprises: a) preparing an inner core layer solution and an outer shell layer solution; b) setting a core flow rate and a shell flow rate, wherein the sum of the core flow rate and the shell flow rate results in a total flow rate; c) electro spraying the microsphere using a set applied voltage; d) collecting the microsphere in a collector bath; and e) exposing the microsphere from step d) to ultraviolet.
[0090] Figure 5 provides another exemplary method as disclosed herein, wherein the method of fabricating the microsphere comprises: a) preparing an inner core layer solution comprising a first cell type, for example, fibroblasts, and an outer shell layer solution comprising a second cell type, for example, keratinocytes; b) setting a core flow rate and a shell flow rate, wherein the sum of the core flow rate and the shell flow rate results in a total flow rate; c) electro spraying the microsphere using an applied voltage; d) collecting the microsphere in (e.g. lOOmM) BaCh; and e) exposing the microsphere from step d) to ultraviolet.
The cell encapsulated microspheres are collected, washed and transferred into cell culture.
[0091] As used in this application, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a genetic marker” includes a plurality of genetic markers, including mixtures and combinations thereof. [0092] As used herein, the terms “increase” and “decrease” refer to the relative alteration of a chosen trait or characteristic in a subset of a population in comparison to the same trait or characteristic as present in the whole population. An increase thus indicates a change on a positive scale, whereas a decrease indicates a change on a negative scale. The term “change”, as used herein, also refers to the difference between a chosen trait or characteristic of an isolated population subset in comparison to the same trait or characteristic in the population as a whole. However, this term is without valuation of the difference seen.
[0093] As used herein, the term “about” in the context of concentration of a substance, size of a substance, length of time, or other stated values means +/- 5% of the stated value, or +/- 4% of the stated value, or +/- 3% of the stated value, or +/- 2% of the stated value, or +/- 1% of the stated value, or +/- 0.5% of the stated value.
[0094] Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
[0095] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention. [0096] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.
[0097] Other embodiments are within the following claims and non- limiting examples. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.
EXPERIMENTAL SECTION
[0098] Material and Methods
[0099] Synthesis of GelMA with Varying Degree of Substitution
[00100] Type A GelMA samples with three different degrees of substitution (DS) were first synthesized. 7.95g of Na2COs and 14.65g of NaHCCh were dissolved in IL of distilled water to prepare 0.25M of carbonate-bicarbonate (CB) buffer solution. Following that, 30g of type A gelatin from porcine skin (gel strength ~175g Bloom, Sigma- Aldrich, St. Louis, MO, USA) was dissolved in 300 mL of the as-prepared 0.25M CB buffer at 50°C. After the gelatin is homogeneously mixed in the CB buffer, the pH value of the gelatin solution was adjusted to 9. Three different volumes of anhydrous methacrylic anhydride (MAA) (94%, Sigma) were separately added to the gelatin solution at a MAA (mL) /gelatin (g) ratio of 0.05, 0.063 and 0.1 mL/g for a target of low, moderate and high DS GelMA under magnetic stirring at 500 rpm. The reaction was left to proceed at 50°C for 3 hours. For the synthesis of low DS GelMA, the reaction was left to proceed without any adjustment of pH whereas for a targeted moderate and high DS GelMA, the pH value of gelatin solution was adjusted to 9 after 30 minutes of MAA addition. Next, IM HC1 was added to the solution and the reaction was stopped when the pH of the solution was reduced to 7.4. The solution was then filtered and dialyzed against DI water at 50°C in a 14kDa dialysis membrane (Membra-Cel™) to remove any unreacted MA and methacrylic acid by-product. The GelMA solution was lyophilized until a dried solid product was obtained and stored at -20°C for future use.
[00101] 'H-N R Confirmation of Methacrylate Substitution in GelMA [00102] The substitution of methacrylate to gelatin was confirmed using proton nuclear magnetic resonance 'H-NMR spectroscopy (Avance Neo 400 MHz, Bruker, Rheinstetten, Germany). 40mg of type A gelatin or GelMA samples were dissolved in 800 pL of deuterium oxide (Sigma) with 0.1 w/v% of trimethylsilylpropanoic acid (TMSP, Sigma) as an internal reference, and the 1 H-NMR-spectra were measured at 40 °C. Using an analytical chemistry software, MestReNova, the phase corrections were applied to all spectra to obtain only the absorptive peaks, baselines were corrected, and the chemical shift scale was adjusted to the TMSP signal at 6(1H) = 0 ppm before the interpretation of results.
[00103] Quantification of GelMA DS
[00104] The different degrees of methacrylate substitution in GelMA samples were quantified by the 2,4,6-Trinitrobenzene sulfonic acid (TNBSA, 5%, Sigma) method. Type A underivatized gelatin and GelMA samples were dissolved in 0. IM CB buffer at a concentration of 900 pg/mL. 500pL of the sample was mixed with 500pL of 0.01% TNBS reagent and incubated for 2 hours at 37°C. The reaction was stopped by adding 250pL of IM HC1 and 500pL of 10% sodium dodecyl sulfate (SDS, Sigma). The absorbance of the samples was measured at 335nm using a SpectraMax M2 microplate reader. The degree of substitution (%) of GelMA can be quantified by comparing the amount of remaining free amino groups in GelMA against that of underivatized gelatin, according to Equation 2 above.
[00105] Rheological Properties of GelMA
[00106] To investigate the effect of shear rate on the viscosity profile of different GelMA - DS solutions, GelMA solutions with different DS were prepared by dissolving the respective GelMA macromers (15%, w/v) in Milli-Q water. The viscosity of the GelMA solutions was then evaluated using a Rheometer MCR 501 (Anton Paar Physica, Graz, Austria) with Anton Paar 25mm cone & plate spindle (CP25-1/TG, gap width=0.047mm). 70pL of the GelMA solution was placed on the platform and the viscosity of GelMA samples was measured under shear rate from 1 to 1000 s 1 at room temperature.
[00107] The mechanical properties of low and high GelMA-DS hydrogels at different GelMA concentrations were characterized via a rheological time- sweep test. Prior to the rheology study, 5%, 10% and 15% (w/v) GelMA solutions of low and high DS were prepared and 0.1% (w/v) of photoinitiator, lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP, Sigma), was added to the GelMA solutions. For the time-sweep measurements, the rheometer is equipped with a glass plate and UV curing system (365nm, 100 mW/cm2). 70pL of the prepared GelMA solutions were then pipetted onto the glass plate and an in- situ photocrosslinking was conducted at room temperature. The frequency of the time-sweep test was set at 1 Hz with a constant shear strain of 3% throughout the entire 6 minutes of the test. After 60s of starting the time- sweep test, the GelMA pre-polymer solutions were irradiated with UV and were crosslinked for 5 minutes before the test stops.
[00108] Microstructure and Porosity of GelMA Hydrogels
[00109] The porous micro structure of GelMA hydrogels with different DS and concentration was investigated using a thermionic scanning electron microscope (SEM, JSM-6360, JEOL, Tokyo, Japan). GelMA macromers with different DS was dissolved in Milli-Q water at different concentration (5%, 10% and 15% (w/v)) and 0.1% of LAP was added to the GelMA solutions. 1 mL of the prepared GelMA solutions were pipetted to a mold and UV-crosslinked for 5 minutes to achieve GelMA hydrogels of different concentrations. Thereafter, the hydrogels were stored at -80°C and lyophilized for 72 hours. Cut cross-sections of the lyophilized GelMA samples were then sputter-coated with gold (JFC-1600, JEOL, Tokyo, Japan) for 120s at 20mA and observed under SEM at an acceleration voltage of 5kV. Using the SEM images obtained, the pore diameter of GelMA hydrogels fabricated under different concentrations was quantified by measuring the longest axes of each pore with the help of ImageJ software. A total of 50 pores were quantified across different SEM images captured at 200x magnification and a pore diameter distribution graph was presented.
[00110] Cell Maintenance and Culture
[00111] To emulate the interplay of keratinocytes and fibroblasts in native cutaneous wound healing, primary human dermal fibroblasts from neonatal foreskins (HDFn, Life Technologies, Carlsbad, CA, USA) and hTERT-immortalized primary neonatal human keratinocytes (kerCTs, ATCC®, CRL4048™, Manassas, Virginia, USA) were cultured for the various studies conducted.
[00112] HDFs were maintained in Dulbecco's modified Eagle's medium (DMEM, Life Technologies) supplemented with 10% fetal bovine serum (FBS, Life Technologies) and 1% of penicillin (100 units/mL) and streptomycin (100 pg/mL) (Life Technologies). kerCTs were maintained in serum-free conditions in keratinocyte basal medium gold (KBM-Gold) (Lonza, Basel, Switzerland) supplemented with KGM-Gold SingleQuots (Lonza) comprising of individual vials of hydrocortisone, transferrin, epinephrine, gentamicin sulfate/amphotericin-B (GA- 1000), bovine pituitary extract, human epidermal growth factor and insulin. The cells were cultured in T150 tissue culture flasks (Corning®, New York, USA) in a 37°C incubator with 5% CO2, with subsequent change in media every 2 days until approximately 80% confluent. Cells passaged 5-9 times were selected for all cell studies.
[00113] MCF7 breast cancer cells and L929 fibroblast cells were maintained in a Dulbecco's modified Eagle's medium (DMEM, Life Technologies) supplemented with 10% fetal bovine serum (FBS, Life Technologies) and 1% of penicillin (100 units/mL) and streptomycin (100 pg/mL) (Life Technologies) at 37°C in the presence of 5% CO2. The cell culture medium is changed every 2 days.
[00114] Fabrication of Cell-laden 3D Bulk GelMA Hydrogel Construct
[00115] For the fabrication of HDFs-laden GelMA-DS40 and kerCTs-laden GelMA- DS90/alginate hydrogels, sterile stock GelMA pre-polymer solutions were first prepared. GelMA-DS40 and GelMA-DS90 macromers were dissolved in phosphate-buffered saline (PBS, Life Technologies) to achieve a stock concentration of 20% (w/v) GelMA-DS40 solution and 25% (w/v) GelMA-DS90 solution respectively. A stock sodium alginate solution was also prepared by dissolving 2% (w/v) sodium alginate (NF, Spectrum Chemical Manufacturing Corp., New Brunswick, USA) in Milli-Q water. The stock solutions were then sterile-filtered through Minisart® polyethersulfone 0.22pm syringe filter (Sartorius, Gottingen, Germany).
[00116] Upon 80% confluency, HDFs and kerCTs were detached from culture flasks using 0.05% Trypsin-EDTA (Life Technologies) and the cells were collected by centrifuging at 1200 rpm for 5 minutes. The cell pellet collected was then re-suspended in PBS (Life Technologies). A fixed volume of cell suspension and 2% sodium alginate (for GelMA/alginate hydrogels) was then mixed with a varying volume of PBS and GelMA stock solutions to achieve a final GelMA concentration of 5%, 10% and 15% GelMA solutions and a final cell concentration of 1x105 cells/mL. Sterile-filtered 0.1% (w/v) LAP crosslinker solution was then added to the cell-laden GelMA pre-polymer solutions. The solutions were homogeneously mixed through pipetting and lOOpL of the cell-laden GelMA solutions were added to a flat bottom Coming® 96 well-plate (n=5 for each group). Photo -polymerization of the samples was then done via UV irradiation (k = 365 nm) for 5 minutes at a fixed height.
[00117] The fabricated cell-laden GelMA hydrogels were then used to study the proliferation of HDFs and kerCTs in different GelMA concentrations of GelMA-DS40 and GelMA- DS90/0.5% alginate hydrogels respectively. [00118] For the fabrication of a microsphere with an cancer cell-core layer and a fibroblastshell layer, MCF-7 cancer cells and L929 fibroblast cells were first detached from culture flasks upon 80% confluency using 0.25% Trypsin-EDTA (Life Technologies) and the cells were collected by centrifuging at 1500 rpm for 5 minutes. The cell pellet collected was then resuspended in PBS (Life Technologies). A fixed volume of cell suspension and 2% sodium alginate (for GelMA/alginate hydrogels) was then mixed with a varying volume of PBS and GelMA stock solutions to achieve a final GelMA concentration of 10% GelMA-DS25 with 10x106 cells/mL MCF-7 cells as the core solution and 10% GelMA-DS96/0.5% alginate with cell concentration of 5x106 cells/mL of L929 cells as the shell solution. The photoinitiator, LAP, was also added to the core and shell solutions to make up into a final concentration 0.1%. [00119] Prior to encapsulation, MCF-7 and L929 cells were stained with cells tracker dye DiO (red) and Dil (green) respectively at a concentration of 1:400 (v/v) in PBS and incubated at 37°C for an hour. The fluorescent-tagged cells were then collected by centrifuging at 1500 rpm for 5 minutes before adding to the core and shell GelMA solutions respectively.
[00120] The core and shell solutions were electro -sprayed into a lOOmM BaC12 collector bath and UV crosslinked for 5mins. Subsequently, the cell-laden core-shell microspheres were rinsed in sodium chloride solution before transferring to a 12-well plate containing DMEM media supplemented with 10% FBS and 1% antibiotic-antimycotic solution.
[00121] Quantification of Proliferation of Cells in 3D GelMA Hydrogels
[00122] The proliferation of HDFs and kerCTs in different concentrations of GelMA-DS40 and GelMA-DS90/0.5% alginate hydrogel constructs was then quantified spectrophotometrically using Cell Counting Kit-8 (CCK8, Dojindo Bio., Japan) according to the manufacturer’s protocol.
[00123] A standard curve correlating known cell numbers to the absorbance value of CCK- 8 at 450nm was first done. HDFs were seeded in a range of IxlO5 cells to 5xl05 cells in 10% GelMA-DS40 hydrogels whereas kerCTs were seeded in a range of IxlO5 cells to 5xl05 cells in 10% GelMA-DS90/0.5% alginate hydrogels. The cell-laden hydrogels were cultured in 200pL of their respective culture medium for 24 hours before replacing with 10% (v/v) CCK- 8 in HDFs or kerCTs culture medium. The samples were then incubated at 37°C for 2 hours in the dark. Thereafter, lOOpL were withdrawn from the contents and transferred to a new 96- well plate. The absorbance of the samples was measured at 450nm using a SpectraMax M2 microplate reader. The absorbance obtained was then plotted against the known number of cells to achieve a linear fit equation for quantification of kerCTs and HDFs in their respective GelMA hydrogels.
[00124] For the cell proliferation study, 10,000 cells of HDFs were seeded in 5%, 10% and 15% GelMA-DS40 hydrogels while 10,000 cells of kerCTs were seeded in 5%, 10% and 15% GelMA-DS90/0.5% alginate hydrogels. Blank GelMA-DS40 and GelMA-DS90/0.5% alginate hydrogels without encapsulation of cells were also fabricated at different GelMA concentrations.
[00125] 200pL of HDFs and kerCTs culture medium were added to the respective blank and cell-laden GelMA hydrogel constructs. The samples were cultured over 7 days and media were changed every 2 days. The proliferation of HDFs and kerCTs in their respective GelMA hydrogels was then evaluated following 1, 3, 5 and 7 days of culture. At each time point, the cell culture medium was aspirated and replaced with 200pL of 10% (v/v) CCK-8 in HDFs or kerCTs culture medium. The samples were incubated at 37 °C for 2 hours in the dark. Thereafter, lOOpL were withdrawn from the contents and transferred to a new clean 96-well plate. The absorbance of the samples was measured at 450nm using a SpectraMax M2 microplate reader and the cell proliferation over days were analysed.
[00126] A net absorbance value of cells in each concentration of GelMA hydrogel was obtained by deducting the obtained absorbance value of each cell-laden hydrogel sample with that of its blank hydrogel. The actual cell number was then quantified from the net absorbance value using the linear fit equations obtained from standard curves.
[00127] The growth rate of cells from a specific time point to the next was quantified based on Equation 3, where N(t2) is the number of cells at second time point, and N(tl) is the number of cells at first time point, and the fold change in cell numbers was normalized against time (days). As the growth rate of cells across different time points is of interest in this study (i.e., from day 1 to day 3, day 3 to 5), the fold change in cell numbers was normalized against t=2 days as shown.
Growth rate (cells/day) — Equation 3
Figure imgf000032_0001
[00128] Synthesis of Fluorescein-labeled Alginate
[00129] Fluorescein-labeled alginate was synthesized by conjugating sodium alginate (Spectrum Chemical Manufacturing Corp.) with fluoresceinamine (isomer I, Sigma) via an EDC-NHS coupling reaction. [00130] Briefly, 150 mg of sodium alginate was dissolved in 10.0 mL PBS overnight. Following that, 7.5 mg of fluoresceinamine, 450 mg of N-(3-Dimethylaminopropyl)-N’- ethylcarbodiimide hydrochloride (EDC, Sigma) and 225 mg of N-Hydroxysuccinimide (NHS, Sigma) were added into the alginate solution. The reaction was left to proceed at room temperature for 24 hours under stirring. The solution was then dialyzed against water in a 3500 Da dialysis membrane for 5 days and the water was replaced 2-3 times per day. Finally, the fluorescein-conjugated alginate solution was lyophilized until a dried solid product was obtained and stored in a vacuum desiccator for further use.
[00131] Fabrication of Fluorescein-Labeled Core-Shell Microspheres with Varying Shell Thickness
[00132] As the shell thickness of the core-shell microspheres plays a critical role in the transfer of nutrients and oxygen from the external environment to the encapsulated skin cells, the tunability of shell thickness at varying core-shell flow rates was investigated. To aid in the visualization of the core-shell compartment within the fabricated microspheres, fluorescein- labelled alginate was used in place of sodium alginate as part of the shell solution.
[00133] 10% GelMA-DS40 solution was prepared as the core solution and 10% GelMA- DS90 with 0.5% fluorescein-labelled alginate were prepared as the shell solution. 0.1% LAP was then added to both the core and shell solutions. The prepared core-shell solutions were infused through a 16-21G co-axial nozzle and sprayed at varying core-shell flow rates of 5:10 mL/hr, 2.5:12.5 mL/hr and 1:14 mL/hr respectively, while keeping the total flow rate constant as 15 mL/hr. The microspheres were then collected in a lOOmM BaCh bath and UV- crosslinked for 5 minutes. An inverted fluorescence microscope (1X53 Olympus, Tokyo, Japan) was used to confirm the fabrication of 3D GelMA fluorescein-labelled core-shell microspheres. The morphology of the fluorescein-labeled core-shell microspheres fabricated under varying flow rates was then captured at lOx magnification.
[00134] Visualization of Core-Shell Architecture Using Confocal Microscopy
[00135] The fluorescein-labeled microspheres fabricated under varying flow rates were imaged under an LSM710 confocal microscope (Zeiss, Jena, Germany) to study the effect of varying core-shell flow rates on the shell thickness of core-shell microspheres. Upon excitation at 488nm, the fluorescein-alginate shell will emit green fluorescence and hence allow the visualization of the shell compartment. Orthogonal z-stack images (z=30pm, total of 12 layers) of the fluorescein-labeled core-shell microspheres were then acquired using a 10X objective. [00136] Quantification of Shell Thickness
[00137] To quantify the shell thickness of the microspheres fabricated under varying coreshell flow rates, a cut planar section of the microspheres was captured using a confocal microscope. Through the planar fluorescent images, the shell thickness was determined by normalizing the fluorescence area of the shell (marked by X in Figure 3) against the planar cross-sectional area of the microsphere (7tr2) where r is the radius of the circle. The radius was quantified based on the diameter of the microspheres, as measured using ImageJ software (line Y in Figure 3).
[00138] Using the quantified area of fluorescent shell and diameter of the circle, the different shell thickness of microspheres fabricated under varying flow rates was then calculated as a percentage based on Equation 1 above.
[00139] Indirect Immunofluorescence Staining
[00140] An indirect immunofluorescence staining protocol was adopted for the staining of proliferation marker (ki67), keratinocytes marker (cytokeratin-5, K5) and fibroblasts marker (vimentin) across the different 2D and 3D cell studies.
[00141] Cells were rinsed with pre-warmed PBS twice and fixed with 4% paraformaldehyde (PFA, Sigma) for 15 minutes at room temperature. Subsequently, the cells were permeabilized using 0.1% v/v Triton™ X-100 (Sigma) and blocked with 3% (v/v) normal goat serum (Abeam, Cambridge, UK) in PBS for an hour at room temperature. After blocking, cells were incubated overnight with diluted primary antibodies at 4°C. Primary antibodies targeting ki67, K5 and vimentin were diluted in blocking buffer at the following concentrations listed in Table 1.
[00142] Table 1. List of primary antibodies diluted in blocking buffer at optimized concentration used in the various studies.
Figure imgf000034_0001
[00143] After overnight incubation with primary antibodies, cells were washed with PBS thrice, for 5 minutes each. Thereafter, fluorochrome-conjugated secondary antibodies and a nuclear staining dye, Hoechst 33342, were added to the samples and left at room temperature for an hour. The fluorochrome-conjugated secondary antibodies targeting mouse and rabbit antibodies were diluted in the blocking buffer at the following concentrations listed in Table 2. Hoechst 33342 dye was also added to the same blocking buffer solution at a concentration of 1:1000. The immunofluorescence- stained cells were washed with PBS thrice for 5 minutes each, before analyzing under a fluorescence microscope.
[00144] Table 2. List of secondary antibodies diluted in blocking buffer at the optimized concentration used.
Figure imgf000035_0001
[00145] Immunofluorescence Staining
[00146] Keratinocytes and fibroblasts are of different origins and are characterized according to their specific phenotypic markers. Cytokeratin-5 (K5), a type II intermediate filament protein, is primarily expressed in basal keratinocytes of the epithelial cells while vimentin, a type III intermediate filament, constitute the major cytoskeletal component of mesenchymal cells such as fibroblasts.
[00147] Upon encapsulation in 3D GelMA core-shell microspheres, kerCTs and HDFs were co-cultured over 7 days in serum-free co-culture media and the media was changed every 2 days. To minimize the loss of microspheres during the staining process, where several washing steps were involved, the cell-laden microspheres were cultured in 24-well 8pm transwell inserts (n=3 wells for each time point). The cellular growth of HDFs and kerCTs in their respective compartment was investigated following 1, 3, 5 and 7 days of co-culture.
[00148] At a specific time -point, the cells were fixed and stained with a cocktail primary antibody containing anti-K5 and anti- vimentin antibodies. After overnight incubation of primary antibodies at 4°C, a secondary antibody solution containing goat anti-mouse Alexa Fluor 594, goat anti-rabbit Alexa Fluor 488, and Hoechst dye were then used to stain the cells. Thereafter, the stained cells were imaged under an LSM710 confocal microscope with a 10X objective. Z-stack images of the compartmentalized cells were acquired (z=15 pm, average of ~21 layers depending on height of each microsphere) and presented as a maximum projection image across the different days. Images depicting the morphological growth of the two different cells in their core-shell compartment at the different axis (x-, y- and z-) were presented as 3D renderings.
[00149] Violet (Vex = 405nm), blue (Vex = 488nm), and green (Vex = 561nm) lasers were employed in the confocal microscope to analyze the cell nuclei, vimentin-expressing HDFs and K5-expressing kerCTs respectively. Transmitted light DIC images of the corresponding cellladen microspheres were captured along with the fluorescence images through the use of a TPMT detector in the confocal microscope.
[00150] Viability of Co-Cultured Skin Cells in 3D GelMA Core-Shell Microspheres
[00151] The cell viability of kerCTs-HDFs encapsulated in 3D GelMA core-shell microspheres was studied using Live/Dead® Viability Assay Kit (Life Technologies) consisting of 4 mM Calcein AM and 2 mM Ethidium homodimer- 1 (EthD-1). A live-dead solution was first prepared by diluting the Calcein AM and EthD-1 to a final concentration of 2pM and 4pM respectively in a serum-free co-culture media.
[00152] kerCTs and HDFs encapsulated in GelMA core-shell microspheres were co-cultured over 7 days in serum-free co-culture media and the media was changed every 2 days. For ease of handling and to allow a uniform change in media, the cell-laden microspheres were cultured in 100pm cell strainers placed in a 6-well plate (n=3 wells for each time point). The cell viability was investigated following 1, 3, 5 and 7 days of co-culture.
[00153] To quantify the viability of co-cultured skin cells in GelMA core-shell microspheres, the microspheres-containing cell strainers were first removed from the well plate at each time point and placed into a new 6-well plate. The strainers were inverted and a fixed volume of the live-dead staining solution was pipetted over the strainers to transfer the cell-laden microspheres into the new well plate. The microspheres were then incubated in the live-dead solution for 30 minutes at 37°C. This step is carried out to reduce the amount of live-dead solution required, as staining the cell-laden microspheres along with the large cell strainers would require about 5 mL of live-dead solution for each sample. Thereafter, the stained cellladen microspheres were filtered and collected using a new cell strainer. The cell strainer now containing stained cell-laden microspheres were thoroughly washed with pre-warmed PBS three times and subsequently placed into a well plate containing 0.25% Trypsin-EDTA with enough volume (~5 mL) to fully cover the microspheres. The well plate with strainers was then incubated at 37°C for 5 minutes for the digestion of microspheres to take place. Thereafter, the digestion was stopped by adding a complete medium to the well plate and collecting the digested cell suspension into a 15 mL centrifuge tube. A cell pellet was obtained after centrifuging at 1200 rpm for 5 minutes. The supernatant was aspirated and the cell pellet was gently washed with PBS before centrifuging the cell suspension.
[00154] Finally, the cell pellet was homogenously resuspended in PBS and analyzed under a 3-channel fluorescence cell counter (NanoEnTek Arthur™, Seoul, Korea). Live and dead cells were counted based on expressing cells in the green channel (kex = 458 ± 20nm) and red channel (kex = 530 ± 20 nm) respectively. To remove any background signal, unstained singlecell suspension was analyzed using the fluorescence cell counter. Threshold and gating parameters were set using unstained cells suspension and samples from different time points were gated according to the same set of parameters.
[00155] The cell viability was then quantified by normalizing the number of live cells by the total number of cells and expressed as a cell viability percentage based on Equation 4.
Figure imgf000037_0001
Cell viability (%) =
Figure imgf000037_0002
x 100% Equation 1
[00156] A qualitative study on the viability of co-cultured skin cells in core-shell microspheres was also conducted to corroborate the viability quantification. At each time point, the 100pm cell strainers were removed from the well plate and placed into a new well plate. The strainers were inverted and a fixed volume of the live-dead staining solution was pipetted over the strainers to collect the cell-laden microspheres into the new well plate. The microspheres were then incubated in the live-dead solution for 30 minutes at 37°C. Thereafter, the stained cell-laden microspheres were imaged under LSM710 confocal microscope with a 10X dry objective. Z-stack images of the encapsulated live-dead cells were acquired (z=15 pm, total of 24 layers) and presented as a maximum projection image across the different time points.
[00157] Proliferation of Keratinocytes in Mono-Cultured and Co-Cultured System
[00158] The proliferation of kerCTs when mono-cultured and co-cultured with HDFs in 3D core-shell microspheres were evaluated by staining single cells suspension with proliferation marker ki67, and keratinocyte phenotypic marker, K5, followed by a cell quantification using Arthur™ Fluorescence Cell Counter. [00159] For the cell proliferation study, kerCTs were mono-cultured or co-cultured with HDFs in GelMA core-shell microspheres over 7 days. The same serum-free co-culture media was used to culture both groups and the media was changed every 2 days. The cell-laden microspheres were cultured in 100pm cell strainers and placed in a 6-well plate (n=3 wells for each time point) to ensure the volume of media accessed by the cells is uniform across the different samples. The proliferation of kerCTs in monoculture and co-culture systems was then investigated following 1, 3, 5 and 7 days of culture.
[00160] Briefly, the cell strainers containing cell-laden microspheres were removed from the well plate at each time point and washed twice with pre-warmed PBS. The strainers were subsequently placed into a well plate containing 0.25% Trypsin-EDTA with enough volume to fully cover the microspheres and incubated at 37 °C for 5 minutes for the microspheres to be digested. Thereafter, the digestion was stopped by adding a complete medium to the well plate and collecting the digested cell suspension into a 15 mL centrifuge tube. A cell pellet was obtained after centrifuging at 1200 rpm for 5 minutes. The supernatant was aspirated and the cell pellet was gently resuspended with PBS before transferring to a 2 mL Eppendorf tube. The Eppendorf tubes were then centrifuged at 15x100 g for 5 minutes in a microcentrifuge (Pico™ 21, Life Technologies). The obtained cell pellet was fixed and stained with primary antibodies solution containing anti-ki67 and anti-K5 antibodies. The cells suspension was then incubated with the primary antibodies overnight at 4°C and thereafter, a secondary antibody solution containing goat anti-mouse Alexa Fluor 488 and goat anti-rabbit Alexa Fluor 594 antibodies were used to stain the cells. The cell suspension was washed with PBS twice before analyzing in Arthur™ fluorescence cell counter.
[00161] To set the gating parameters, both unstained kerCTs and K5 -stained kerCTs were used. kerCTs cultured in 2D culture flasks at 80% confluence were trypsinized and the cell suspension was stained with primary antibody, anti-K5, followed by secondary antibody Alexa Fluor 488. Both unstained and K5-antibody stained kerCTs were then analyzed under the Arthur™ fluorescence cell counter. The cells were first gated for green fluorescent protein (GFP) and red fluorescent protein (RFP) signals based on the background signal from the unstained cells. The GFP signals which correspond to K5 -expressing cells (kerCTs) were then gated such that all the analyzed cells were included for quantification within the gating parameter, i.e., 100% of K5-positive cells. This is because all the cells analyzed should express K5 as they are all keratinocytes. [00162] Cells digested from GelMA core-shell microspheres were then gated using the same parameters obtained. The percentage of proliferative kerCTs in monoculture and co-culture systems were then quantified based on Equation 5, where cells expressing both ki67 and K5 (indicating proliferative kerCTs) were normalized against the total number of cells expressing K5 (total number of kerCTs).
Proliferative keratinocytes (%) = Cells exPressing both ki67 and K5 x ]()()% Equation 5
All cells expressing K5
[00163 ] Statistical Analysis
[00164] All the experimental results reported in the studies were expressed as mean ± standard deviation of at least three replicates (n=3). Statistical significance between different study groups was analyzed using one-way analysis of variance (ANOVA) and followed-up with a posteriori Tukey’s honestly significant difference (HSD) test. All statistical analysis were done using OriginPro software and significant differences were measured at p<0.05.
[00165] Co-delivery and Attachment of Proliferative Keratinocytes and Fibroblasts
[00166] An immunofluorescence staining of the released cells from 3D GelMA core-shell microspheres was done following 7 and 14 days of culture to confirm the co-delivery and attachment of HDFs and kerCTs. To determine if the kerCTs and HDFs cells were able to retain their proliferative capacity after delivery from GelMA core-shell microspheres, the proliferative state of the delivered cells was also studied through immunofluorescence staining of proliferation marker, ki67 and fibroblasts marker, vimentin. Anti-vimentin antibody, a marker for fibroblasts, were used instead of keratinocytes marker, anti-K5 antibody, for the study on the proliferative capacity of the delivered cells. This is to allow easier visualization of the elongated HDFs cytoskeleton and could give more information about the growth of HDFs, which otherwise, the HDFs would appear as just individual nuclei without the vimentin staining,
[00167] Upon encapsulation, cells-laden GelMA core-shell microspheres were cultured in a 6 well-plate to provide a large enough surface area for the investigation of cells delivery and growth over 7 and 14 days. The cell-laden microspheres were cultured in the skin co-culture media and a fixed volume of media were changed every 2 days across the different samples. Care was taken to minimize the removal of microspheres during every change in media.
[00168] On days 7 and 14, the samples (n=6 wells for each time point) were washed with pre-warmed PBS and stained with primary antibodies. To characterize and investigate the attachment and growth of kerCTs and HDFs after delivery, the cells were stained with cocktail primary antibodies containing anti-K5 and anti-vimentin antibodies, which are markers for kerCTs and HDFs respectively. To evaluate if the delivered kerCTs and HDFs were able to retain their proliferative capacity after delivery, a cocktail of primary antibodies containing anti-ki67 and anti-vimentin antibodies were used to stain the cells. After overnight incubation of the respective cocktail primary antibodies in 4°C, a secondary antibody solution containing goat anti-mouse Alexa Fluor 594, goat anti-rabbit Alexa Fluor 488, and Hoechst dye were then used to stain the cells for both studies. Thereafter, the stained cells were imaged under an inverted fluorescence microscope at 4x, lOx and 20x magnifications.
[00169] Cell Viability of Delivered Keratinocytes and Fibroblasts
[00170] The viability of cells delivered from 3D GelMA core-shell microspheres was studied after 7 days of culture through the Live/Dead® Viability Assay Kit (Life Technologies) consisting of 4 mM Calcein AM and 2 mM Ethidium homodimer- 1 (EthD-1). A live-dead solution was first prepared by diluting the Calcein AM and EthD-1 to a final concentration of 2pM and 4pM respectively in a serum-free co-culture media.
[00171] Upon encapsulation, cells-laden GelMA core-shell microspheres were cultured in a 6 well-plate in co-culture media (n=3 wells). A fixed volume of media was changed every 2 days across the different samples and the viability of delivered cells following 7 days of culture was studied. The co-culture media was aspirated as much as possible while ensuring minimal uptake of the cell-laden microspheres. A fixed volume of the live-dead solution was added to each well and samples were incubated in the live-dead solution for 30 minutes at 37°C. Thereafter, the stained cell-laden microspheres, as well as the delivered cells, were imaged under an inverted fluorescence microscope at 4x magnifications.
[00172] Fabrication of 3D GelMA core-shell microspheres
[00173] In the co-axial electro spraying process, the GelMA core and shell solutions containing 0.1% of crosslinker LAP were loaded into two different syringes and infused through a co-axial needle with the help of syringe pumps. At an applied voltage, the core-shell solutions were electro- sprayed into microspheres and collected in a collector bath. The collected core-shell microspheres were subsequently UV-crosslinked under 365nm at a fixed height (2 petri-dish). An illustration on the experimental setup is shown in Figure 4.
[00174] To investigate the optimal conditions in fabricating GelMA core-shell microspheres with a narrow size distribution, several parameters were studied: GelMA core and shell concentration (5% and 10%, w/v), applied voltage (7.5-12 kV), type of collector bath (lOOmM of BaC12 and CaC12), total core-shell flow rate (9-19 mL/hr) and co-axial needle size (18-14G and 21-16G). During the study, the distance between the tip of the nozzle and the grounded electrode was kept constant at 5 cm. Each process parameter was optimized, while keeping the others constant. The morphology of fabricated GelMA core-shell microspheres was examined using an inverted optical microscope (1X53 Olympus, Tokyo, Japan). To analyze the size distribution of microspheres fabricated under different parameters, 50 microspheres were randomly imaged and analyzed using ImageJ software. 200 random microspheres were then imaged to obtain the size distribution of core-shell microspheres fabricated under the optimized parameters.
[00175] Fabrication of 3D Cell-laden GelMA core-shell microspheres
[00176] GelMA-DS40 and GelMA-DS90 macromers were dissolved in phosphate-buffered saline (PBS, Life Technologies) to achieve a stock concentration of 20% (w/v) GelMA-DS40 solution and 20% (w/v) GelMA-DS90 solution respectively. A stock sodium alginate solution was also prepared by dissolving 2% (w/v) sodium alginate (NF, Spectrum Chemical Manufacturing Corp., New Brunswick, USA) in Milli-Q water. The stock solutions were then sterile-filtered through Minisart® polyethersulfone 0.22pm syringe filter (Sartorius, Gottingen, Germany).
[00177] Upon 80% confluency, HDFs and kerCTs were detached from culture flasks using 0.05% Trypsin-EDTA (Life Technologies) and the cells were collected by centrifuging at 1200 rpm for 5 minutes. The cell pellet collected was then re-suspended in PBS (Life Technologies). A fixed volume of cell suspension and 2% sodium alginate (for GelMA/alginate hydrogels) was then mixed with a varying volume of PBS and GelMA stock solutions to achieve a final GelMA concentration of 10% GelMA-DS40 with 4xl06 cells/mL HDFs and 10% GelMA - DS90/0.5% alginate with cell concentration of 20xl06 cells/mL kerCTs.
[00178] 4xl06 cells/mL of HDFs-laden 10% GelMA-DS40 solution was prepared as the core solution and 20xl06 cells/mL of kerCTs -laden 10% GelMA-DS90-0.5% alginate solution was prepared as the shell solution. 0.1% (w/v) of sterile photo-initiator, lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP, Sigma), was added to both core and shell solution prior to spraying.
[00179] The core-shell solutions with two different cells were infused through a co-axial nozzle of 16-21G at a core-shell flow rate of 2.5: 12.5 mL/hr. Under an applied voltage of 9kV, the two solutions were sprayed into core-shell microspheres encapsulating HDFs and kerCTs in their respective core and shell compartment. The microspheres were collected in a lOOmM BaCh collector bath and UV-crosslinked under 365nm for 5 minutes at a fixed height. The fully crosslinked cell-laden GelMA core-shell microspheres were then filtered through a 100pm cell strainer and washed thoroughly with a 150mM NaCl solution to remove any excess BaCh that may harm the cells (ionic displacement of Ba2+ by Na+). The filtered cell-laden GelMA core-shell microspheres were then transferred to a 15 mL centrifuge tube by pipetting the skin co-culture media onto the cell strainer. The cell-laden GelMA core-shell microspheres were resuspended in the co-culture media before aliquoting an equal volume for different cell studies. An illustration of the experimental setup in fabricating the cell-laden GelMA core-shell microspheres is shown in Figure 5.
[00180] Co-culture of Skin Cell-laden GelMA Core-Shell microspheres
[00181] Upon fabrication of cell-laden core-shell microspheres, the microspheres were cultured in a chemically defined serum-free skin co-culture media (Atlantis Bioscience, Singapore). To ensure the volume of media accessed by the cells is uniform across the samples and minimize the loss of microspheres during media change, the cell-laden microspheres were cultured in 100pm cell strainers (Sigma).
[00182] Experimental Results
[00183] Synthesis of GelMA with Varying Degree of Substitution
[00184] Type A gelatin methacrylate (GelMA) was first fabricated via a facile one -pot synthesis between Type A gelatin and methacrylic anhydride (MAA) as described in the methodology section. By adjusting the volume of MAA added to type A gelatin (0.05mL, 0.063mL and O.lmL of MAA per gram of gelatin), GelMA with three different degrees of methacryloyl substitution can be obtained. The substitution of MAA to gelatin molecules was then confirmed by XH-NMR analysis (Figure 6).
[00185] The successful conjugation of methacrylamide grafts to gelatin was confirmed by the presence of new peaks in contrast to that of pristine gelatin at 8 = 5.4 -5.7 ppm (region A) which corresponds to the acrylic protons (2H) in methacrylamide grafts. As the feed ratio of MAA to gelatin increases from 0.05/1 to 0.1/1 mL/g, the intensity of the peaks at region A increases, indicating a higher degree of substitution of methacrylamide grafts to gelatin. Conversely, the increment of MAA feed ratio results in a decreasing signal at 6 = 2.8 - 3.0 ppm (region B), which corresponds to the methylene protons of unreacted lysine. As the reaction between MAA and gelatin occurs at the lysine binding site through nucleophilic substitution, the corresponding drop in the lysine methylene peak intensity as MAA feed ratio increases indicated that more lysine groups had reacted with the MAA and there was an increased methacryloylation with lysine groups. The ’ H-NMR spectra thus confirm the successful conjugation of methacrylamide grafts to gelatin, resulting in the synthesis of gelatin methacrylate (GelMA) with an increasing degree of methacryloyl substitutions as the MAA/gelatin feed ratio increases.
[00186] The DS of three different batches of GelMA samples fabricated under MAA/gelatin feed ratio of 0.05/1, 0.063/1 and 0.1/1 (mL/g) were quantified and an average DS across the three different GelMA batches was then presented as shown in Figure 7. The increase in feed ratio of MAA to gelatin showed an increasing degree of substitution and the degree of substitution was found to be proportional to the feed ratio of MAA, with a two-fold increase in the degree of substitution as the feed ratio of MAA/gelatin increases from 0.05/1 to 0.1/1 (mL/g). From the TNBSA assay quantification, the three different DS of GelMA fabricated were found to be 41.73%, 57.10% and 90.74%. For ease of labeling, the different DS was rounded to their nearest tens and labeled as GelMA DS40, DS60 and DS90 for subsequent experiments as shown in Table 3.
[00187] Table 3. Summary of GelMA with varying DS.
Figure imgf000043_0001
[00188] Rheological Properties of GelMA Hydrogels with Different DS for Fabrication of Core-Shell Microspheres
[00189] In order to form a core- shell structure, mixing between the core and shell solutions must be minimized when both solutions are in contact with each other at the tip of the co-axial nozzle. A large dissimilarity in viscosity between the core and shell solution is therefore desired.
[00190] The viscoelastic behavior of GelMA during the spraying process can be determined by investigating the effect of shear rate on the viscosity of GelMA solutions with varying DS at 25°C. The GelMA concentration was fixed at 15% (w/v) across the different solutions.
[00191] It can be seen from the viscosity profile (Figure 8) that all three different GelMA- DS solutions exhibited a shear-thinning behavior at 25°C, where the viscosity decreases with increasing shear rate. The pseudo-plastic property of GelMA solutions demonstrated the ability of GelMA solutions to flow through the co-axial nozzle of the spray system without clogging. It was also shown that GelMA with a higher DS exhibit a lower viscosity. This is attributed to the higher degree of methacryloyation of free amino groups or hydroxyl groups in gelatin chains which reduces the interchain or intrachain hydrogen bonding present in higher DS of GelMA and hence resulting in lesser resistance to flow (lesser viscosity). From the viscosity profile, GelMA-DS40 and DS90 solutions exhibited the largest difference in viscosity across the different shear rates, which results in a higher probability they do not mix during the spraying process. GelMA-DS40 and GelMA-DS90 are therefore selected as exemplary candidates for the core- shell solutions.
[00192] From the time- sweep experiment, GelMA DS 90 hydrogel was found to exhibit a significantly higher storage modulus as compared to GelMA DS40, after 5 minutes of photocrosslinking. GelMA-DS90 is thus selected as the shell solution for the fabrication of coreshell microspheres so that the stiffer, higher crosslinked shell can protect the softer GelMA - DS40 core after spraying and UV-crosslinked into microspheres. To facilitate the formation of microspheres, 0.5% of sodium alginate was also added to the GelMA-DS90 shell solution to allow a quick crosslinking upon collection in a divalent cationic collector bath.
[00193] GelMA concentration on porosity of hydrogel
[00194] The cross-sectional SEM micrographs reveal the honeycomb structure of the GelMA-DS40 and DS90 hydrogels fabricated under different GelMA concentrations (Figure 9 A). The honeycomb structure is critical for the adhesion of cells while facilitating the diffusion of nutrients, oxygen, and waste exchange to the encapsulated cells.
[00195] It was shown that the pore sizes of GelMA-DS40 and GelMA-DS90 hydrogels decrease with increasing GelMA concentrations due to the higher crosslinking density (Figure 9B). The average pore size of GelMA-DS40 hydrogels was found to decrease from 64.73pm in 5% GelMA to 24.05pm in 10% GelMA and then to 13.93pm in 15% GelMA-DS40, as seen from the shift of the normal curve fitted on the pore diameter distribution (Figure 9B (left)) across the different GelMA concentration. For GelMA-DS90 hydrogels, the average pore size was found to decrease from 38.30pm in 5% GelMA-DS90 to 36.83pm in 10% GelMA-DS90 and then to 27.45pm in 15% GelMA-DS90, as seen from the shift of the normal curve fitted on the pore diameter distribution (Figure 9B (right)) across the different GelMA concentration.
[00196] GelMA concentration on mechanical properties of GelMA hydrogels [00197] GelMA concentration also has an effect on the stiffness of GelMA hydrogels. As shown in Figures 10A and 10B, the storage modulus of GelMA hydrogels increased with an increase in GelMA concentrations for both low and high GelMA-DS. The average storage modulus of GelMA hydrogels at the end of the 5 minutes of UV-crosslinking was quantified and presented in Figure 10C, which demonstrated that increasing GelMA concentration from 5%, 10% to 15% increases the storage modulus of hydrogels for both GelMA-DS. This is due to the increased availability of cross-linkable methacryloyl groups as GelMA concentration increases, resulting in higher crosslinking density as seen from the SEM micrographs in Figure 9A. It can also be seen from the time-sweep test that while 10% and 15% GelMA-DS40 and DS90 hydrogels were able to rapidly crosslink and reached a plateau after the GelMA solutions were UV-irradiated (fully crosslinked), this was not the case for 5% GelMA-DS40 and 5% GelMA-DS90. A gradual increase in storage modulus was observed, although at a much later time for 5% GelMA-DS40, and both hydrogels did not exhibit any plateau in storage modulus.
[00198] GelMA concentration on cell growth within 3D bulk GelMA hydrogels
[00199] It was found that the stiffness of GelMA hydrogels increased with increasing GelMA concentrations for both low and high GelMA-DS, owing to the increased availability of crosslinkable methacryloyl groups as GelMA concentration increases, resulting in higher crosslinking density. In order to fabricate a mechanically stable GelMA core-shell microspheres that can support the encapsulated cell growth, cell growth was observed within GelMA hydrogels of varying concentration.
[00200] To emulate the architecture of the native skin, where fibroblasts (dermis) is located underneath the keratinocytes in the stratified epidermis, primary neonatal human dermal fibroblasts (HDFs) will be encapsulated in the core (GelMA-DS40) whereas hTERT- immortalized primary neonatal human keratinocytes (kerCTs) will be encapsulated in the shell (GelMA-DS90/0.5% alginate) of the core-shell microspheres.
[00201] HDFs suspension was then mixed with GelMA-DS40 solutions of different concentrations at a cell density of IxlO5 cells/mL and the cell-laden GelMA prepolymer solutions were UV cross-linked for 5 minutes. HDFs-laden GelMA hydrogels were then cultured over 7 days in their culture medium and the cell proliferation was quantified using CCK-8 assay.
[00202] The proliferation study in Figure 11A demonstrated that all concentrations of GelMA-DS40 hydrogels were able to support the growth of HDFs over 7 days, as seen by the increase in cell numbers for all groups after 3, 5 and 7 days of culture. HDFs cultured in 5% GelMA DS40 hydrogel was found to have proliferated more than 10% and 15% for all time points, whereas those cultured in 10% GelMA-DS40 proliferated better than 15% GelMA- DS40.
[00203] The growth rate of HDFs over 7 days of culture were calculated based on Equation 3 and analyzed as shown in Figure 1 IB. It was shown that the growth rate of HDFs from day 1 to day 3 of culture was the highest in 5% GelMA-DS40 as compared to the other two groups and was higher than those cultured in 15% GelMA-DS40. After 3 days of culture, the relative growth rate of HDFs in all concentrations of GelMA-DS40 hydrogels dropped even though the cell number were shown to be increasing. This could be attributed to the confluence of HDFs within the hydrogels after 3 days of culture as well as the transition of HDF cells from the exponential growth phase to the stationary phase.
[00204] The growth of kerCTs in GelMA-DS90/0.5% alginate hydrogel with different GelMA concentrations was also investigated. kerCTs were encapsulated in GelMA- DS90/0.5% alginate solutions of different GelMA concentrations at a cell density of IxlO5 cells/mL and the cell-laden GelMA prepolymer solutions were UV cross-linked for 5 minutes. kerCTs-laden GelMA hydrogels were then cultured over 7 days in their culture medium and the cell proliferation was quantified. The proliferation study in Figure 11C demonstrated that all concentrations of GelMA-DS90 hydrogels, even in the presence of 0.5% alginate, were able to support the growth of kerCTs over 7 days, as seen by the increase in cell numbers for all groups after 3, 5 and 7 days of culture.
[00205] From the proliferation study, kerCTs cultured in 5% GelMA DS90/0.5% alginate hydrogel was found to have proliferated more than 10% and 15% for all time points. The study on the growth rate of kerCTs over 7 days of culture further supported this, where the growth rate of kerCTs from day 1 to day 3 of culture was the highest in 5% GelMA-DS90/0.5% alginate as compared to the other two groups and was higher than those cultured in 15% GelMA-DS90/0.5% alginate. While the growth rate of all groups subsequently reduces after 3 days of culture due to the inherent growth behavior of cells to transit from exponential growth to stationary phase over time, the growth rate of kerCTs was higher in 15% GelMA-DS90 as compared to 5% GelMA-90 from day 3 to day 5 of culture. This is because kerCTs cultured in 5% GelMA-DS90/0.5% alginate could have reached confluence after 3 days of culture whereas the less confluent kerCTs in 15% GelMA-DS90/0.5% alginate (due to lesser growth from day 1 to day 3) had more available substrate space to grow. The confluency of keratinocytes plays a critical role in its cell proliferation due to the nature of keratinocytes to grow as colonies and undergo cell-contact inhibition upon confluency.
[00206] Thickness of shell layer
[00207] The thickness of the shell layer plays an important role in the transfer of nutrients and oxygen from the external environment to the encapsulated skin cells. In circumstances where the shell layer is too thick, it will limit the diffusion of nutrients and waste exchange to the encapsulated cells, especially those in the core layer of the core-shell. This may result in a hypoxic core where the lack of oxygen will reduce the cell viability and capability to proliferate.
[00208] To visualize the core-shell architecture, alginate, a component of the shell material, was first conjugated with a fluorescent labelling reagent, aminofluorescein. The fluorescein isothiocyanate (FITC)-labeled alginate was then used to fabricate core-shell microspheres at 3 different core-shell flow rates of 5:10 mL/hr, 2.5: 12.5 mL/hr andl:14 mL/hr respectively.
[00209] A distinct core-shell structure was observed via the orthogonal z-stack images and the shell thickness of the microspheres was shown to increase with increasing flow rate of shell solution (Figure 12). The average shell thickness of the microspheres was found to increase linearly with the increase in flow rate of shell solution (Figure 13). Increasing the flow rate of shell solution from 10 mL/hr to 14 mL/hr (while keeping the total flow rate as 15 mL/hr) saw a three-fold increase in the shell thickness, from 30% to 93% of its total cross-sectional area. In addition, the relationship between shell thickness and shell flow rate can be modeled, for example, according to a linear fit equation derived from the quantified data, allowing the estimation of the shell thickness in microspheres fabricated at any specific core-shell flow rate. [00210] As the keratinocytes and fibroblasts are co-cultured in a 5:1 ratio with the keratinocytes encapsulated in the shell and fibroblasts in the core, the shell thickness of microspheres should be thick enough to encapsulate the five-fold higher of keratinocytes relative to the fibroblasts but at the same time, the shell thickness should allow sufficient oxygen and nutrient exchange between the external environment and the encapsulated cells.
[00211] Fabricating the core-shell microspheres at a core-shell flow rate of 1:14 mL/hr produced microspheres with large shell thickness, making up 93% of its total area. This shell thickness would be too thick for efficient diffusion and exchange of nutrients, oxygen and waste between the external environment and encapsulated cells, especially so for the fibroblasts encapsulated in the core of the microspheres. Conversely, fabricating at a core-shell flow rate of 5:10 mL/hr produced core-shell microspheres with very thin shell that makes up only 30% of its total area. A shell too thin will greatly limit the space for the high concentrated keratinocytes to grow in which may then compromise on the viability of the keratinocytes.
[00212] The core-shell flow rate of 2.5:12.5 mL/hr was found to be able to fabricate microspheres with an optimal shell thickness of 70% of its total area, a shell thick enough to encapsulate the high-density keratinocytes while having a relatively larger core compartment for oxygen and nutrient to diffuse more efficiently to the encapsulated fibroblasts.
[00213] The use of co-axial electrospray system renders the capability to fine-tune the thickness of the shell layer just by controlling the core-shell flow rate and allows the encapsulation of two different skin cells in their desired co-culture ratio. The optimal shell thickness could provide a biological environment that supports the growth of the encapsulated skin cells and provide for the recapitulation of the keratinocytes-fibroblasts crosstalk in the native skin.
[00214] Cell densities in core and shell layer
[00215] The propensity of mono-cultured fibroblasts to grow in the core of the fabricated microspheres was then studied over 7 days. HDFs-laden GelMA solutions with varying cell densities of IxlO6 cells/mL, 3xl06 cells/mL and 4xl06 cells/mL were first sprayed and the growth of mono-cultured HDFs encapsulated in the core of microspheres was studied by brightfield imaging.
[00216] The initial HDFs seeding densities of IxlO6 cells/mL and 3xl06 cells/mL were found to be too low as only a few fibroblasts cell was found to be encapsulated in the core of the microspheres (Figure 14). This resulted in sparse growth of HDFs for both groups over 7 days. Spraying at a higher cell density of 4xl06 cells/mL enabled the encapsulation of a higher number of HDFs in the core, whereby after 7 days of culturing, the cells aggregated and extended to form an interconnected network surrounding the core of the microspheres. The close proximity of HDFs with each other is required to promote autocrine signals essential for cell survival, as seen by the difference in growth between HDFs encapsulated in low density (3xl06 cells/mL) and high density (4xl06 cells/mL).
[00217] 4xl06 cells/mL of HDFs-laden GelMA solution was therefore prepared as the core solution, and considering the 1:5 co-culture ratio of fibroblasts to keratinocytes in recapitulating the epidermal-mesenchymal interactions in full-thickness skin equivalent, 20xl06 cells/mL of kerCTs-laden GelMA-alginate solution was prepared as the shell solution. HDFs and kerCTs were then encapsulated in the core-shell microspheres by co-axial spraying of the core and shell solutions.
[00218] GelMA core-shell microspheres as a ‘mini-bioreactor’ for patient-derived cells of interest
[00219] The GelMA-alginate core-shell microspheres were found to be fully digested by incubating the microspheres in 0.25% trypsin-EDTA for 5 minutes which is a typical cell dissociation method used in cell culture. This mild treatment with 0.25% trypsin-EDTA allows the GelMA-based microspheres to be readily degraded and release the cells without compromising on the cell viability or intracellular functions.
[00220] Trypsin is a type of serine protease that cleave peptide chains at the carboxyl side of positively charged amino acids such as lysine or arginine. The specificity of trypsin in cleaving peptide chains of positively-charged amino acids is due to the presence of negatively-charged aspartate residue in the catalytic pocket (SI) of trypsin which attracts and stabilizes the positively charged amino acids. As the gelatin backbone in GelMA contains cell-binding RGD motifs (Arg-Gly-Asp peptides) along with other free amine groups such as lysine and alanine, trypsin will cleave the peptide bonds of the positively charged arginine and lysine present in gelatin, allowing the digestion of GelMA microspheres.
[00221] In addition, 0.25% trypsin-EDTA (Life Technologies) contains 380 mg/L of hydrated ethylenediaminetetraacetic acid (EDTA), which is a metal ion chelating agent. EDTA readily chelates with a metal ion and forms a stable bond between the nitrogen atom of EDTA and the metal ion. The shell of GelMA core-shell microspheres consists of 0.5% alginate which upon contact with BaCh collection bath, crosslinks with barium ions to form barium alginate. The introduction of trypsin-EDTA during digestion thus chelates with the barium metal ions present in barium alginate, causing the crosslinked alginate to be dissolved.
[00222] It is therefore envisioned that patient-derived cells of interest can be encapsulated into the relevant core or shell layer of the GelMA microspheres. The GelMA microspheres can act like a ‘mini -bioreactor’, allowing the encapsulated cells to undergo cell expansion, and thereafter, before the GelMA microspheres are intentionally degraded with trypsin to release the cells for tissue regeneration purposes.
[00223] Evaluation of co-cultured cell viability in GelMA core-shell microspheres [00224] The cell viability of co-cultured keratinocytes-fibroblasts in GelMA core-shell microspheres was evaluated 1, 3, 5 and 7 days after culture (Figure 15). The co-cultured skin cells in GelMA core-shell microspheres were found to be highly viable at 74 ± 5.57% after 1 day of culture and maintained at 76 ± 3.21% after 3 days of culture, with no significant difference between the two time points. This indicates that the porous microstructure of GelMA core-shell microspheres provided the encapsulated skin cells a viable biological environment to grow in, despite spraying the cells at a high voltage of 9kV. The high cell viability could also be attributed to the RGD motifs present in GelMA which facilitates cell adhesion upon encapsulation in the microgel. However, after culturing the cell-laden microspheres for 5 days, the viability of the skin cells decreased to 44 ± 0.57% (p< 0.05) and sustained at 40 ± 9.84% after 7 days of culture.
[00225] This is corroborated by Figure 16 A, which shows that the GelMA microspheres contained mostly viable cells on day 1 and 3 of culture. Past 5 and 7 days of culture, the number of dead cells increases, indicating a loss of cell viability.
[00226] The decrease in viability of encapsulated skin cells decreased from day 3 to day 5 of co-culture could be attributed to an overcrowding of cells as the cells populate over days in a compact microenvironment, causing the cells to experience a high competition for oxygen and nutrients with each other. As the kerCTs were encapsulated in a 5-fold higher density than HDFs, it is of interest to determine the distribution of live and dead cells within the core and shell compartment of the GelMA core-shell microspheres. Following 3 days of co-culture, the encapsulated skin cells exhibited high number of calcein-expressing live cells throughout the GelMA core-shell microspheres shown in Figure 16B, indicating that both the HDFs and kerCTs encapsulated in the core and shell compartments of the microspheres were highly viable.
[00227] Notably, following 5 days of co-culture, a higher number of dead cells expressing EthD-1 were present in areas of confluent cells and along the periphery of the core-shell microspheres (Figure 16C). The effect of confluency and overcrowding could have played a bigger role in the apoptosis of kerCTs as compared to the HDFs encapsulated in the core. The 5-fold higher density of kerCTs encapsulated in the shell of the microspheres, as well as the cell-cell contact inhibition (of proliferation) induced due to the continual expansion and confluency of kerCTs over time, could be the reasons leading to a higher cell death observed in the periphery of the core- shell micro spheres. [00228] Next, the viability of heterotypic cells co-cultured in core-shell microspheres was investigated. 10xl06 cells/ml MCF-7 cancer cells were encapsulated in the core of the microsphere, and 5xl06 cells/ml L929 fibroblasts cells were encapsulated in shell of the microsphere. A live-dead staining using calcein-AM and Ethidium Homodimer- 1 (EthD-1) showed that the co-cultured cells are still viable after encapsulating in the microspheres post 24 hours. The co-cultured cells were also observed to proliferate over 3 days as seen by the increase in number of Vybrant DiO (ThermoFisher) labelled MCF-7 cells in the core and CellTracker™ CM-Dil (ThermoFisher) labelled E929 cells in the shell (Figure 32).
[00229] Morphological Growth of Co-Cultured Skin Cells in GelMA Core-Shell Microspheres
[00230] Upon encapsulation of HDFs and kerCTs in core-shell microspheres, the skin cells- laden microspheres were cultured over 7 days and their morphology were characterized using microscopy imaging (Figure 22A). The cells were observed to be randomly distributed as single cells in the transparent GelMA microspheres right after encapsulation (day 0) as indicated by the black arrow, but aggregated to form clusters of cells within the microspheres following 7 days of culture (white arrow). Notably, GelMA microspheres began to degrade on day 7 of culture and the encapsulated HDFs and kerCTs were released. This degradation of the microsphere observed is a natural biological process due to the biodegradable feature of GelMA.
[00231] As a control, mono-cultured kerCTs were encapsulated in the shell of core-shell microspheres without fibroblasts to evaluate the morphological difference between monocultured kerCTs and co-cultured skin cells in GelMA core-shell microspheres over 7 days of culture (Figure 22B). An apparent empty core was observed (black arrow) in the shell of the core-shell microspheres. Upon encapsulation, the mono-cultured kerCTs were randomly distributed as single cells within the shell and grew as colonies following 7 days of culture (white arrow), indicating that the growth of kerCTs was supported within the shell of the GelMA core-shell microspheres. However, the cells did not aggregate to form cell clusters over the 7 days of culture as observed when co-cultured with fibroblasts (indicated by the white arrow in Figure 22 A).
[00232] The aggregated cell cluster observed in co-cultured HDFs-kerCTs could be the interconnected network of HDFs surrounding the core of core-shell microspheres, which is confirmed by immunostaining kerCTs and HDFs as shown in Figure 22C. The vimentin- expressing HDFs formed a small interconnected network in the core of the core-shell microspheres after 1 day of culture, and the cluster of HDF cells grew in size over the days. Following 7 days of culture, HDFs have elongated along the x-y and z- dimensions and proliferated to form a large 3D interconnected network that surrounds the core compartment of the core-shell microspheres.
[00233] The single-cell k5-expressing kerCTs encapsulated in the shell of microsphere grown from a single cell on day 1 of culture to clusters of colonies following 7 days of coculture. This finding is consistent with the brightfield images captured for mono-cultured kerCTs in Figure 22B which depicted the growth of kerCTs from single cells to colonies following 7 days of culture.
[00234] This confirmed that 3D GelMA core-shell microspheres are able to support the growth of encapsulated HDFs and kerCTs over 7 days of co-culture, wherein the two inherently different fibroblasts -keratinocyte cell types were able to expand within their respective coreshell compartment. The fluorescent images further suggest that there was no obvious HDFs contamination into the shell of the GelMA microspheres and mixing with the encapsulated kerCTs. The GelMA core-shell microsphere thus allows the compartmentalized HDFs-kerCTs to grow at their own rate. This prevents the potential problem of overgrowth of fibroblasts and space with keratinocytes, which occurs when both keratinocytes and fibroblasts are typically cultured within the same system.
[00235] Delivery and Characterization of Co-cultured Cells from 3D Core-Shell Microspheres
[00236] It was demonstrated that co-culturing of kerCTs with HDFs enhanced the proliferation of kerCTs in comparison to mono-cultured kerCTs without HDFs. As keratinocytes promote wound closure, it would be essential for the cell-laden GelMA coreshell microspheres to degrade and release keratinocytes for wound management. It was observed that the cell-laden GelMA core-shell microspheres began to degrade after 7 days of culture and the encapsulated cells were delivered, or released.
[00237] Upon delivery, mono-cultured kerCTs continued to proliferate and formed small patches of cell colonies after 7 days of culture, as indicated by the white arrow in Figure 23C. The colonies of “cobblestone” cells, which are morphological characteristics of keratinocytes, were also observed in the co-cultured kerCTs shown in Figure 23F as indicated by the white arrow. Similarly, the colonies of “cobblestone” cells were also observed in the co-cultured kerCTs and HDFs. In addition, elongated spindle-shaped cells, which are indicative of HDFs, were observed alongside the “cobblestone” cell colonies in the co-cultures. The morphological difference between the larger and more elongated cells (black arrow) as compared to the colonies of smaller ‘cobblestone’ cells (white arrow) in Figure 23F indicated that HDFs were successfully co-delivered with kerCTs after 7 days of co-culture.
[00238] Both HDFs and kerCTs were able to attach onto the well plate and proliferated to form a small patch of skin cells. The released kerCTs attached to the well plate upon delivery and proliferated and migrated to form small sheets of K5 expressing keratinocytes as seen in Figures 24E and 24F. Vimentin-expressing HDFs were also successfully delivered from the core-shell microspheres seen in Figures 24G and 24H, albeit in lesser numbers than kerCTs. This is due to the encapsulation of kerCTs in the shell of the core-shell microspheres which allow the keratinocytes to be released first before HDFs could. The HDFs released similarly attached to the well plate and grew along the sheet of kerCTs as single cells, indicated by white arrows in Figures 24H.
[00239] The immunofluorescence staining of an attached core-shell microsphere along with the delivered cells in Figure 25 showed that there were vimentin-expressing cells in the microsphere as indicated by the white arrow in the magnified image (Figures 25B and 25E). A single channel depicting the 3D network of encapsulated HDFs in the core of the microsphere was observed in Figure 25E. The images corroborated that following 7 days of co-culture, while kerCTs were released from the shell compartment, the majority of the HDFs was still encapsulated in the core of the GelMA core-shell microspheres which explains for the lesser number of HDFs delivered as shown in the previous figure (Figure 24).
[00240] The small patches of kerCTs colonies seen on day 7 of culture were observed to have proliferated and migrated further to form a large epithelial sheet following 14 days of coculture, as indicated by the white arrows in the brightfield images captured (Figure 26A). More HDFs were found to have been delivered by day 14 and have proliferated and grown into an elongated sheet of fibroblasts (indicated by the black arrows) with a distinct edge that lines along the colonies of kerCTs. The growth of HDFs into a dermal layer is critical in the re- epithelization of wounds as it allows the mimicry of ECM properties in the native human skin by providing structural integrity to the epithelial sheet, elasticity and a vascular bed.
[00241] As a control, the growth of delivered kerCTs when mono-cultured in 3D GelMA core-shell microspheres after 14 days were also studied. Like the co-culture system, the mono- cultured kerCTs were found to have proliferated to form a large epithelial sheet as shown in Figure 26B. There was no distinct lining of elongated cell sheets observed within the colonies of keratinocytes which indicate the absence of HDFs.
[00242] The successful co-delivery of kerCTs and HDFs from degraded GelMA core-shell microspheres was confirmed by immunofluorescence staining following 14 days of co-culture. The delivered skin cells were found to express both k5 and vimentin markers which indicate the presence of kerCTs and HDFs respectively (Figure 27). A large epithelial sheet of kerCTs with HDFs elongating along the edge of the epithelial sheet was observed through the phase contrast and fluorescence images. The area enclosed by the white dotted line in the vimentin staining (Figure 27, bottom right) showed the propensity of delivered HDFs to align and elongate along the edge of the kerCT colonies. The results demonstrated the ability of the codelivered HDFs and kerCTs to form a large sheet of skin cells after 14 days of co-culture, which can be used for wound coverage.
[00243] Cell Viability of Delivered Skin Cells
[00244] The viability of the delivered kerCTs and HDFs were then qualitatively evaluated. Following 7 days of co-culture, cells released from the GelMA core-shell microspheres were stained with live-dead dye (calcein-AM/ethidium homodimer- 1) and representative images of two different samples were then captured using a fluorescence microscope as shown in Figures 33A-B.
[00245] The delivered kerCTs and HDFs were observed to be viable after 7 days of co- culture. Almost all the delivered cells were stained green (live) and only a few red fluorescent cells (dead) were observed as shown in Figure 33. This finding was similar across all the 3 different samples. The high cell death seen in the GelMA core-shell microspheres is similar to the previous study done on the cell viability of encapsulated skin cells, which showed that only 40 ± 9.84% of cells were viable after 7 days of co-culture due to space constraint within the microspheres. Therefore, while it was demonstrated that the skin cells were able to attach and expand into a viable sheet of skin cells upon delivery from 3D GelMA core-shell microspheres, the timing of delivery could be shortened to avoid the high cell death observed in the cell-laden GelMA core-shell microspheres at the later stage of culture.
[00246] Proliferative State of Delivered Cells
[00247] Following 7 days of co-culture, the delivered kerCTs were shown to retain their proliferative capacity, as indicated by the ki-67 positive cells without vimentin staining (Figure 28). Notably, only a few HDFs were found to be proliferative, as indicated by the cells expressing both ki67 and vimentin. The observed proliferative state of HDFs and kerCTs are due to their inherent difference in cell growth rate.
[00248] Following 14 days of co-culture, the delivered kerCTs were proliferative, as shown by the ki67-positive, non-vimentin expressing cells (Figure 29). This indicated that the proliferative capacity of kerCTs was maintained a week later from when it was delivered on day 7 of culture, when the cells have proliferated to form a confluent sheet of keratinocytes. Similar to day 7 findings, the co-delivered HDFs were found to be proliferative.
[00249] Cells expressing both ki67 and K5 (indicating proliferative keratinocytes) were normalized against the total number of cells expressing K5 (total number of keratinocytes) and expressed as a percentage in Figure 30. An approximate 4-fold increase in ki67-positive keratinocytes was observed when co-cultured with fibroblasts, as compared to its mono-culture group following 1 day of culture. This is attributed to the paracrine crosstalk between fibroblasts and keratinocytes, which are in close proximity to each other when encapsulated in the core-shell microspheres. Following 3 days of co-culture with fibroblasts, the percentage of ki67-positive keratinocytes decreased and was lower than its mono-cultured counterpart. While there was no significant drop in ki67 -positive keratinocytes for the subsequent days of coculturing with fibroblasts, the percentage of ki-67 positive keratinocytes remained lesser than that of mono-cultured keratinocytes. This is not surprising because the high percentage (about 84%) of proliferative keratinocytes after 1 day of co-culture meant that the keratinocytes were expanding in numbers and there was not much space left for the keratinocytes to continue to expand after day 1 of co-culture. The reduction in the percentage of ki67-positive keratinocytes after a spiked increase was also observed in mono-cultured keratinocytes, where the number of proliferative keratinocytes significantly increase after day 3 of culture but declined on the days that followed, as presented in the line graph in Figure 30. Although a decrease in ki67-positive keratinocytes was observed in both mono- and co-culture after the spiked increase, co-culturing keratinocytes with fibroblasts actually enhanced the proliferation of keratinocytes as compared to mono-cultured keratinocytes. This shows the potential that these 3D GelMA core-shell microspheres can co-deliver the keratinocytes and fibroblasts for enhanced wound healing in a facile manner.
[00250] The results highlight the capability of delivering proliferative co-culture cells from 3D GelMA core-shell microspheres in a facile, high yield manner. For example, if keratinocytes and fibroblasts used in the shell and core layers of the microsphere, the delivered cells are able to attach and expand into a proliferative skin construct for extensive wound management. The capability to deliver both kerCTs and HDFs as well as the growth of the delivered skin cells into a large epidermal-dermal layer is critical in the re-epithelization of wounds; as the proliferative keratinocytes would help in the acceleration of wound closure while the dermal layer allows the recapitulation of functionality in the regenerated skin, providing structural integrity to the epithelial sheet, elasticity and a vascular bed.
[00251] Influence of Processing Parameters on Core-Shell Microspheres
[00252] The interplay of electro spraying parameters such as applied voltage, collector bath and core- shell feed rate on the morphology and size of fabricated GelMA core- shell microspheres will then be studied in this section. Herein, the electro spraying distance was kept constant at 5cm as the distance between the nozzle and collector bath is inversely proportional to the strength of the electrostatic field (shorter distance, stronger electric field).
[00253] To fabricate core-shell microspheres that can support the encapsulated cell growth, core-shell solutions consisting of 5% GelMA-DS40 and 5% GelMA-DS90/0.5% alginate, respectively, were first electro- sprayed at different applied voltages shown in Figure 17. The core-shell solutions were sprayed using 21-16G co-axial needle size and at a total flow rate of 15 mL/hr. The fabricated microspheres were collected in a lOOmM BaC12 collector bath and UV-crosslinked for 5 minutes.
[00254] It was observed that spraying 5% GelMA-DS40 and 5% GelMA-DS90/0.5% alginate as the core-shell solutions at 7.5kV formed microspheres with a distinct elongated ‘tail’ structure. This indicates that 5% GelMA-DS40 and 5% GelMA-DS90/0.5% alginate were too low a concentration for sufficient crosslinking to support the structural integrity of the fabricated core-shell microspheres. In addition, reducing the voltage to less than 7.5kV was found to fall below the critical voltage where the stable cone-jet mode transited to dripping mode and microspheres could not be sprayed. Increasing the voltage to 9kV and above results in the breakage of microspheres.
[00255] The concentration of shell solution was then increased to 10% GelMA-DS90/0.5% alginate and was found to form ovoid-shaped particles at 7.5kV and 9kV. While a better morphology of microspheres was observed when spayed at 10.5kV, the microspheres had a large variation in size (Figure 18). A more monodispersed microspheres were achieved when sprayed with an optimal core-shell concentration of 10% GelMA-DS40 and 10% GelMA- DS90/0.5% alginate, at an applied voltage of 9kV.
[00256] The critical range of the electric field in which the stable cone -jet mode is formed is also dependent on the concentration of GelMA core- shell solutions. As the concentration of GelMA decreases, its viscosity decreases due to the lesser hindrance from the bulky MA side groups and hence lesser resistance to flow. As such, with the decrease in GelMA concentration, the lower viscosity and entanglement of GelMA polymer chains allows the GelMA molecular chains to move more freely, resulting in the deformation of the GelMA core-shell droplet at the tip of the nozzle before the voltage was applied. Therefore, other than the standalone effect that GelMA concentration has on the stiffness of GelMA hydrogels to maintain the structural integrity of the fabricated core-shell microspheres after UV-crosslinking, the GelMA concentration also affects the stability of GelMA core-shell droplet at the nozzle tip which subsequently influences the critical range of electric field that can be applied to achieve a stable cone-jet mode.
[00257] Calcium chloride is a divalent ion that can crosslink with alginate for the fabrication of microspheres and is considered food-safe. It was therefore investigated as an alternative to BaCh as the collection bath. From Figure 19A, it was observed that microspheres collected in lOOmM CaCh were not able to form uniform spherical particles and had a wider size distribution as compared to those collected in BaCh. This is due to the lower ionic strength of Ca2+ ions resulting in a weaker interaction between the cations and the negative charges in the alginate chains, and hence less stable core-shell microspheres.
[00258] Next, the effects on the morphology and size of the core-shell microspheres was investigated when using a larger co-axial needle (18-14G) (Figure 19A). Spraying of core-shell microspheres using a larger co-axial needle size of 18-16G was able to fabricate smaller microspheres but with a wider size distribution. Therefore, in the pursuance of achieving a uniform size distribution of core-shell microspheres, a co-axial needle size of 21-16G and collector bath of lOOmM BaCh solution should be used.
[00259] In addition, the influence of total feed rate on the morphology and polydispersity of core-shell microspheres was studied. While it was observed that the size of microspheres was reduced with decreasing total flow rate, the size distribution was found to be wider. A lower flow rate allows the applied electric field to disperse the jet stream more evenly and thoroughly into smaller droplets but too low a flow rate is not fast enough to form a stable core- shell droplet at the exit of the nozzle before being dispersed by the electric field. Spraying at a total flow rate of 15 mL/hr were able to fabricate core-shell microspheres with the least poly disperse size distribution (Figure 19B).
[00260] Using the parameters as disclosed above, 3D core-shell microspheres were fabricated with a nearly monodisperse size distribution of 395pm ± 25pm (Figure 20).
[00261] High yield and scalability of core-shell microspheres
[00262] To determine the scalability of electro spraying GelMA core-shell microspheres, the microspheres were fabricated at a core-shell flow rate of 2.5:12.5 mL/hr respectively and sprayed for a total of 3 minutes. 0.125ml of cells-laden core solution and 0.625 ml of cells- laden shell solution was tested. Thereafter, the microspheres were collected and transferred to a 15 mL centrifuge tube with phenol-red DMEM culture media and then to a Nunc® 90mm petri dish (Figure 21A). The number of microspheres was quantified based on coverage.
[00263] An ImageJ analysis on the area covered by the fluorescent GelMA core-shell microspheres (Figure 21B) showed that the area covered was 40cm2, which is approximately 70% of the Nunc® 90mm petri dish with a surface area size of 58cm2 (as listed on the product details by Sigma).
[00264] This is a promising aspect in the delivery of HDFs and kerCTs to a massive wound, whereby spraying for 3 minutes requires only 125pL of 4xl06 cells/mL of HDFs-laden GelMA and 625pL of 20xl06 cell/mL of keratinocytes -laden GelMA solution. With such small volumes of cells and materials required, it is envisioned that the fabricated skin cell-laden GelMA core-shell microspheres will be able to cover 70% of a 90mm diameter wound before releasing the encapsulated proliferative skin cells in a clinical setting. Furthermore, all of the pre-polymeric solutions are infused through the co-axial nozzle were sprayed into core-shell microspheres on the co-axial electrospray platform, allowing a 100% yield from the materials used. This would minimize material wastage and allows for an efficient, low cost and highly scalable fabrication of cell-laden GelMA core-shell microspheres as a one-step co-delivery system.
[00265] In addition, the use of the co-axial electrospray thus brings forth several other advantages; the most notable advantage is that different cells can be encapsulated in the coreshell microspheres in a facile manner, as any additional procedures will reduce the viability of the encapsulated cells. [00266] The independent flow of two different liquids prior to spraying means high molecular weight polymers can be used as long as its viscosity allows it to flow at working temperature and more critically, there is no surfactant involved in the fabrication of core-shell microspheres which if present, would be detrimental for the cells. Other advantageous property of co-axial electrospray includes its scalability, reproducibility and ease of handling from the simple experimental set-up to the collection of core-shell microspheres. Unlike microfluidics which requires expertise and time to design, fabricate and optimise the device, co-axial electrospray proved to be a much simpler platform for the fabrication of scalable core-shell microspheres.
[00267] The present invention creates a highly versatile microsphere that encapsulates and co-delivers active ingredients such as cells, drugs or proteins in a facile, low-cost and high- yield manner, which renders it useful in a myriad of applications. When cells are encapsulated, the microsphere has a microenvironment that enables the encapsulated cells to retain their biological activity. As the microsphere has tunable properties, it would be possible to control the rate of release of active ingredients such as cells, drugs or proteins depending on the need of the subject.
[00268] Using the method presented, it is envisioned that hundreds to thousands of miniaturized 3D tissues can be fabricated in a facile manner by electrospraying different active ingredients such as cells, drugs or proteins in the core-shell microspheres, which are then delivered to a subject in need. The method as disclosed herein is valuable for mass production of cells to facilitate translation of the emerging cell-based regenerative medicine into the clinic, which cannot be achieved by the conventional technique of using low -attachment culture plates. For example, by encapsulating and delivering high yield of co-cultured human keratinocytes and fibroblasts in microspheres, it can enhance the re-epithelisation of large wounds in patients. For a different application, two or more different types of drugs, with or without cells, can be encapsulated simultaneously in the core-shell microspheres for systemic drug delivery.

Claims

58
1. A microsphere comprising: an inner core layer comprising gelatin methacryloyl (GelMA), wherein the inner core layer further comprises a first cell type; and an outer shell layer comprising gelatin methacryloyl (GelMA) and alginate, wherein the outer shell layer comprises a second cell type.
2. The microsphere of claim 1, wherein the thickness of the outer shell layer is about 5- 99% of the microsphere.
3. The microsphere of claim 1 or 2, wherein the thickness of the outer shell layer is about 70% of the microsphere.
4. The microsphere of any one of claims 1-3, wherein the outer shell layer is stiffer than the inner core layer.
5. The micro sphere of any one of claims 1-4, wherein the GelMA of the inner core layer comprises a degree of methacryloyl substitution (DS) of about 35%-60%.
6. The micro sphere of any one of claims 1-5, wherein the GelMA of the outer shell layer comprises a degree of methacryloyl substitution (DS) of about 65%-96%.
7. The microsphere of any one of claims 1-6, wherein the inner core layer and outer shell layer each comprise a concentration of about 5-15% (w/v) GelMA.
8. The microsphere of any one of claims 1-6, wherein the outer shell layer comprises about 0.1 -1.0% alginate.
9. The microsphere of any one of claims 1-8, wherein the first cell type or second cell type comprises anchorage dependent cells, non-anchorage dependent cells, or a combination thereof. 59 The microsphere of any one of claims 1-9, wherein the first cell type is selected from a group consisting of fibroblasts, cancer cells, keratinocytes and stem cells. The microsphere of claim 10, wherein the first cell type is fibroblasts. The microsphere of any one of claims 1-11, wherein the second cell type is selected from a group consisting of keratinocytes, fibroblasts, epithelial cells, cancer cells, endothelial cells, and stem cells. The microsphere of claim 12, wherein the second cell type is keratinocytes. The microsphere of any one of claims 1-13, wherein the inner core layer comprises fibroblasts and the outer shell layer comprises keratinocytes. The microsphere of any one of claims 10-14, wherein the inner core layer comprises IxlO6 cells/ml to 4xl06 cells/ml fibroblasts. The microsphere of claim 12-14, wherein the outer shell layer comprises 5xl06 cells/ml to 20x106 cells/ml keratinocytes. A synthetic skin replacement manufactured from the microsphere of any one of claims 14-16. A method of promoting wound healing comprising administering the microsphere of any one of claims 1-16 or a synthetic skin replacement of claim 17 to a subject in need thereof. A method of tissue regeneration comprising administering the microsphere of any one of claims 1-16 or a synthetic skin replacement of claim 17 to a subject in need thereof. A method of treating a disease comprising administering the microsphere of any one of claims 1-16 or a synthetic skin replacement of claim 17 in a subject in need thereof. 60 The method of claim 20, wherein the disease is a skin disease or disorder. The method of claim 21, wherein the skin disease or disorder comprises bum injury, Recessive Dystrophic Epidermolysis Bullosa, diabetic foot ulcers, infectious wounds, ischemic wounds, open wounds, and chronic wound. The method of any one of claims 18-22, wherein the administration comprises topical, subcutaneous, intravenous, or intramuscular administration. A method of fabricating the microsphere of any one of claims 1-16, the method comprising: a) preparing an inner core layer solution and an outer shell layer solution; b) setting a core flow rate and a shell flow rate, wherein the sum of the core flow rate and the shell flow rate results in a total flow rate of 9-19 ml/hr; c) electro spraying the microsphere. The method of claim 24, wherein the total flow rate of 15 ml/hr. The method of claim 24, wherein the core flow rate is about 2.5 ml/hr and the shell flow rate is about 12.5ml/hr. The method of any one of claims 24-26, wherein a co-axial nozzle is used for electro spraying in step c). The method of claim 27, wherein the co-axial nozzle is 16-21G or 18-14G. The method of any one of claims 24-28, wherein an applied voltage of 7.5 - 12kV is set for electro spraying. The method of any one of claims 24-29, further comprising: d. collecting the micro sphere in BaCh or CaCh. 61
31. The method of claim 30, further comprising: e. exposing the microspheres from step d) to ultraviolet.
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