WO2022154754A1 - Microsphères du type cœur-écorce - Google Patents

Microsphères du type cœur-écorce Download PDF

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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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PCT/SG2022/050011
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English (en)
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Lay Poh Tan
Pei Leng TAN
Huizhi CHEN
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Nanyang Technological University
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Publication of WO2022154754A1 publication Critical patent/WO2022154754A1/fr

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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

La présente invention concerne une microsphère avec une couche de cœur interne comprenant un premier type de cellule et une couche d'écorce externe comprenant un second type de cellule, et son utilisation. Dans un mode de réalisation, le cœur interne comprend de la gélatine méthacryloyle (GelMA) ; et la couche d'écorce externe comprend de la gélatine méthacryloyle (GelMA) et de l'alginate. Dans un autre mode de réalisation, le premier type de cellule est des fibroblastes et le second type de cellule est des kératinocytes. La présente invention concerne également le procédé de fabrication de la microsphère.
PCT/SG2022/050011 2021-01-15 2022-01-14 Microsphères du type cœur-écorce WO2022154754A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117462517A (zh) * 2023-11-21 2024-01-30 广州逸芯生命科学有限公司 一种基于微流控技术的高载量水凝胶缓释微球及制备方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018027023A1 (fr) * 2016-08-03 2018-02-08 Wake Forest University Health Sciences Plates-formes de modélisation du cancer et procédés pour leur utilisation
WO2019152767A1 (fr) * 2018-02-02 2019-08-08 Wake Forest University Health Sciences Organoïdes associés à une immunothérapie et leurs procédés de préparation et d'utilisation
CN110339393A (zh) * 2019-07-19 2019-10-18 吉林大学 一种基于水凝胶-核壳微球的创伤敷料及其制备方法
WO2020111503A1 (fr) * 2018-11-28 2020-06-04 포항공과대학교 산학협력단 Structure type cœur-écorce pour établir un micro-environnement d'organoïde normal et cancéreux et son procédé de fabrication

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018027023A1 (fr) * 2016-08-03 2018-02-08 Wake Forest University Health Sciences Plates-formes de modélisation du cancer et procédés pour leur utilisation
WO2019152767A1 (fr) * 2018-02-02 2019-08-08 Wake Forest University Health Sciences Organoïdes associés à une immunothérapie et leurs procédés de préparation et d'utilisation
WO2020111503A1 (fr) * 2018-11-28 2020-06-04 포항공과대학교 산학협력단 Structure type cœur-écorce pour établir un micro-environnement d'organoïde normal et cancéreux et son procédé de fabrication
CN110339393A (zh) * 2019-07-19 2019-10-18 吉林大学 一种基于水凝胶-核壳微球的创伤敷料及其制备方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHEN HUIZHI, LAY POH TAN: "793. Versatile Core-Shell Microgels for Cell Encapsulation in Three-Dimensional Cell Culture", BIOMATERIALS, 6 April 2019 (2019-04-06), XP055956640, [retrieved on 20220831] *
WANG HUI, LIU HAITAO, LIU HUI, SU WENTAO, CHEN WENWEN, QIN JIANHUA: "One‐Step Generation of Core–Shell Gelatin Methacrylate (GelMA) Microgels Using a Droplet Microfluidic System", ADVANCED MATERIALS TECHNOLOGIES, WILEY, DE, vol. 4, no. 6, 11 June 2019 (2019-06-11), DE , pages 1800632, XP055956643, ISSN: 2365-709X, DOI: 10.1002/admt.201800632 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117462517A (zh) * 2023-11-21 2024-01-30 广州逸芯生命科学有限公司 一种基于微流控技术的高载量水凝胶缓释微球及制备方法
CN117462517B (zh) * 2023-11-21 2024-04-30 广州逸芯生命科学有限公司 一种基于微流控技术的高载量水凝胶缓释微球及制备方法

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