US20190142998A1 - Scaffolds fabricated from electrospun decellularized extracellular matrix - Google Patents

Scaffolds fabricated from electrospun decellularized extracellular matrix Download PDF

Info

Publication number
US20190142998A1
US20190142998A1 US16/092,787 US201716092787A US2019142998A1 US 20190142998 A1 US20190142998 A1 US 20190142998A1 US 201716092787 A US201716092787 A US 201716092787A US 2019142998 A1 US2019142998 A1 US 2019142998A1
Authority
US
United States
Prior art keywords
scaffold
poly
ecm
tissue
decellularized
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US16/092,787
Other languages
English (en)
Inventor
Marcelle Machluf
Eyal Zussman
Limor Baruch
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Technion Research and Development Foundation Ltd
Original Assignee
Technion Research and Development Foundation Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Technion Research and Development Foundation Ltd filed Critical Technion Research and Development Foundation Ltd
Assigned to TECHNION RESEARCH & DEVELOPMENT FOUNDATION LIMITED reassignment TECHNION RESEARCH & DEVELOPMENT FOUNDATION LIMITED ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: MACHLUF, MARCELLE, BARUCH, Limor, ZUSSMAN, EYAL
Publication of US20190142998A1 publication Critical patent/US20190142998A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived 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
    • A61L27/14—Macromolecular materials
    • A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/24—Collagen
    • 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
    • A61L27/3604—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 characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A61L27/3633—Extracellular matrix [ECM]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

  • the present invention in some embodiments thereof, relates the use of electrospun decellularized extracellular matrix for fabricating scaffolds.
  • Injured organs or tissues can be replaced via whole organ transplantation.
  • major obstacles limit the surgical procedures used for this purpose, including shortage of donors and the need to use immunosuppressive drugs to prevent rejection of the implanted organ.
  • Tissue engineering has emerged as a promising approach to improve or restore the function or shape of a damaged tissue or organ by implantation of polymeric scaffolds, functional cells, or their combination in cell seeded scaffolds. The success of a scaffold for tissue engineering depends on the manufacturing design, the material chosen, and on the accurate understanding of the scaffold's desired characteristics.
  • ECM extracellular matrix
  • ECM extracellular matrix
  • a scaffold generated according to the method described herein.
  • a scaffold comprising electrospun decellularized ECM of an organ, wherein the to decellularized ECM has a similar protein composition to native ECM of the organ.
  • a scaffold comprising electrospun decellularized ECM, wherein the decellularized ECM is derived from an organ selected from the group consisting of heart and pancreas.
  • composition of matter comprising the scaffold described herein and cells seeded on the scaffold.
  • a method of treating a medical condition which may benefit from cell transplantation in a subject in need thereof comprising transplanting the scaffold described herein into the subject, thereby treating the medical condition.
  • the method further comprises decellularizing a tissue of a subject prior to generate the decellularized ECM prior to step (a).
  • the method further comprises contacting the solution of decellularized. ECM with a polymer so as to increase the viscoelasticity of the solution following step (a) and prior to step (b).
  • the method further comprises filtering the homogenate of decellularized ECM prior to the electrospinning.
  • the organic solvent is selected from the group consisting of acetone, N,N-dimethylformamide (DMF), diethylformamide, chloroform, methylethylketone, acetic acid, formic acid, ethanol, 1,1,1,3,3,3-hexa fluoro-2-propanol (HFIP), tetrafluoroethanol, dichloromethane (DCM), tetrahydrofuran (THF), trifluoroacetic acid (TFA), camphorsulfonic acid, dimethyl acetamide, isopropyl alcohol (IPA) and mixtures thereof.
  • DMF 1,1,1,3,3,3-hexa fluoro-2-propanol
  • DCM dichloromethane
  • THF tetrahydrofuran
  • THF trifluoroacetic acid
  • camphorsulfonic acid dimethyl acetamide
  • the organic solvent is HFIP.
  • the polymer is a biocompatible polymer.
  • the polymer is a hydrophilic polymer.
  • the polymer is a synthetic polymer.
  • the synthetic polymer is selected from the group consisting of poly(D,L-lactide) poly(urethanes), poly(siloxanes), poly(silicones), poly(ethylene), poly(vinyl pyrrolidone), poly(2-hydroxy ethyl methacrylate), poly(N-vinyl pyrrolidone), poly(methyl methacrylate), polyvinyl alcohol) (PVA), poly acrylic acid), poly(vinyl acetate), polyacrylamide, poly(ethylene-co-vinyl acetate), poly(ethylene glycol), poly(methacrylic acid), polyglycolic acids (PGA), poly(lactide-co-glycolides) (PLGA), nylons, polyamides, polyanhydrides, poly(ethylene-co-vinyl alcohol) (EVOH), polycaprolactone, poly(vinyl acetate), polyvinylhydroxide, poly(ethylene oxide) (PEO), polyorthoesters and mixtures thereof.
  • PVA poly acrylic acid
  • the synthetic polymer is PEO.
  • the amount of the PEO in the solution is between 0.05-1% mass.
  • the decellularized ECM is derived from porcine tissue.
  • the method further comprises removing the polymer following the electrospinning.
  • the decellularized ECM comprises collagen type I and collagen type III.
  • the decellularized ECM is devoid of collagen type VI.
  • the scaffold is devoid of a synthetic polymer.
  • the scaffold has been pre-seeded with cells.
  • the medical condition is a cardiac disease.
  • the medical condition is Diabetes.
  • the scaffold is for use in treating a medical condition which may benefit from cell transplantation.
  • FIGS. 1A-E are photographs of porcine ECM electrospun in: acidified water and combined with PE( )to a mass ratio of (a) 7:1, (h) 8:1, and (c) 14:1. Electrospinning of pcECM dissolved in HFIP (d) without and (e) with 0.1 mass % PEO. Scale bar 100 ⁇ m.
  • FIGS. 3A-B are HR-SEM images of porcine pancreas ECM (ppECM) fibers. Scale bars 200 ⁇ m.
  • FIG. 5 is a graph illustrating fiber diameter distribution.
  • FIG. 6 is a graph illustrating pore size distribution in the electrospun pcECM scaffold.
  • FIGS. 7A-B are photographs of initial wetting/contact angle of the electrospun pcECM scaffold (A), and the wetting/contact angle after 5 min (13).
  • FIG. 8 is a photograph of initial wetting/contact angle of the electrospun ppECM scaffold.
  • FIGS. 9A-B are a graph illustrating the Fourier transform infrared spectroscopy (MR) spectra of the electrospun pcECM scaffold compared to decellularized pcECM (A), and the secondary structure of scaffold proteins determined by Fourier deconvolution of their amid I band (B).
  • MR Fourier transform infrared spectroscopy
  • FIGS. 16A-D are graphs illustrating the relative expressions of ECM remodeling genes by hMSCs grown on the electrospun pcECM scaffold compared to native pcECM.
  • Collagen I A
  • Collagen III B
  • C Matrix metalloproteinase-2 (MMP2) (D).
  • FIG. 17 is a LSFM image of electrospun pcECM scaffold seeded with human induced pluripotent stem cells (hiPSCs) at 3 weeks post seeding.
  • hiPSCs human induced pluripotent stem cells
  • FIGS. 18A-D are images of hiPSCs cultured on pcECM electrospun fibrous scaffolds. SEM images (A-C) and H&E staining (D) at 3 weeks post seeding.
  • FIGS. 19A-C are images of cardiomyocytes cultured on pcECM electrospun fibrous scaffolds.
  • FIGS. 20A-C are images of cardiomyocytes cultured on pcECM electrospun fibrous scaffolds for 3 weeks, stained with Hoechst 33258 (DNA-blue) and antibodies for Connexin-43 (q), sarcomeric ⁇ -actinin-SAA (r), and cardiac troponin 1 (cTn1, s) cardiac markers (green).
  • FIG. 21 is a confocal line scan images showing changes in intracellular Ca 2+ in a Fluo-4 loaded neonatal cardiomyocytes seeded electrospun (ES)-pcECM scaffold 2 weeks post seeding. Whole cell Ca 2+ transient in three different induced pacing frequencies are shown.
  • FIG. 22 is a LSFM image of electrospun pcECM scaffold seeded with hiPSCs that were differentiated into cardiomyocytes (hiPSC-CM) at 3 weeks post seeding.
  • green phalloidin-FITC (Actin)
  • blue Hoechst (DNA).
  • FIG. 23 is a graph illustrating the viability of hiPSC-CMs on pcECM electrospun scaffolds after 1, 7 and 14 days.
  • FIG. 24 is a confocal line scan images showing changes in intracellular Ca 2+ in a Fluo-4 loaded hiPSC-CM seeded electrospun pcECM scaffold 14 days post seeding. Whole cell Ca 2+ transient in 1Hz induced pacing frequency are shown.
  • FIGS. 25A-C are graphs illustrating in vitro immunogenicity studies of pcECM electrospun fibrous scaffolds. Pieces of electrospun pcECM scaffold were used to stimulate RAW macrophages, LPS stimulation was a positive control, and PLGA and non-stimulated cells were negative controls. The level of (A) secreted NO and excreted pro-inflammatory cytokines TNF ⁇ and (C) IL1 ⁇ were evaluated.
  • FIGS. 26A-B are graphs illustrating pro-inflammatory cytokine expression of (A) TNF ⁇ and (B) IL1 ⁇ in inguinal lymph nodes of mice that received a subcutaneous implanted electrospun fibrous scaffold from PLGA (dots) and ECM (stripes).
  • FIGS. 27A-I are graphs illustrating complete blood counts of mice following subcutaneous implantation of electrospun PLGA and electrospun pcECM scaffolds.
  • the present invention in some embodiments thereof, relates to the use of electrospun decellularized extracellular matrix for fabricating scaffolds.
  • ECM extracellular matrix
  • scaffolds from decellularized ECM are difficult to control, or finely tune for desired properties. They lack reproducibility and generally have poor mechanical properties, which limits their use.
  • the present inventors now propose electrospinning decellularized ECM for fabrication of biological scaffolds. This would not only provide cells with the natural environment attributed to the ECM, but also provide control over the structure of the fibrous ECM network, allowing the design of scaffolds with specific properties such as degradability, density and mechanical strength.
  • the present inventors Whilst reducing the present invention to practice, the present inventors generated scaffolds which were fabricated from ECM isolated from a single organ—pancreas or heart.
  • the present inventors show that the cells seeded on the scaffolds had a polypeptide composition similar to the native ECM of the organ from which it was derived—Tables 1-2.
  • the average fiber diameter of the scaffold ranged from 300 to 1500 nm.
  • the inventors further demonstrate that cells seeded on the scaffolds were capable of surviving for at least four weeks ( FIGS. 13, 17, 19 and 23 ). Scaffolds fabricated according to the disclosed methods were shown to be non-immunogenic ( FIGS. 25-27 ).
  • a method of generating a scaffold comprising:
  • decellularized ECM refers to the extracellular matrix which supports tissue organization (e.g., a natural tissue) and underwent a decellularization process (i.e., a removal of all cells from the organ) and is thus completely devoid of any cellular components.
  • the decellularized ECM typically comprises a plurality of polypeptides (e.g. collagens).
  • the decellularized ECM comprises collagen alpha-2(I), collagen alpha-1(III) and collagen alpha-1(I).
  • the decellularized ECM may also comprise collagen alpha-2(IV), collagen alpha-1(V) and collagen alpha-1(II) (or fragments thereof)
  • the amount of collagen alpha-2(I), collagen alpha-1(III) and collagen alpha-1(I) is greater in the decellularized ECM of this aspect of the present invention than collagen alpha-2(IV), collagen alpha-1(V) and collagen alpha-1(II).
  • the decellularized ECM may also comprise smaller amounts of collagen alpha-2(VI), collagen alpha-3(VI) and collagen alpha-1(VI) or collagen alpha-1(IV) (or fragments thereof).
  • the amount of collagen alpha-2(I), collagen alpha-1(III) and collagen alpha-1(I) is greater in the decellularized ECM of this aspect of the present invention than collagen alpha-2(VI), collagen alpha-3(VI) and collagen alpha-1(VI) or collagen alpha-1(IV).
  • cellular components refers to cell membrane components or intracellular components which make up the cell. Examples of cell components include cell structures es., organelles) or molecules comprised in same.
  • Such include, but are not limited to, cell nuclei, nucleic acids, residual nucleic acids (e.g., fragmented nucleic acid sequences), cell membranes and/or residual cell membranes (e.g., fragmented membranes) which are present in cells of the tissue. It will be appreciated that due to the removal of all cellular components from the tissue, such a decellularized matrix cannot induce an immunological response when implanted in a subject.
  • extracellular matrix refers to a complex network of materials produced and secreted by the cells of the tissue into the to surrounding extracellular space and/or medium and which typically together with the cells of the tissue impart the tissue its mechanical and structural properties.
  • the ECM includes fibrous elements (particularly collagen, elastin, or reticulin), cell adhesion polypeptides (e.g., fibronectin, laminin and adhesive glycoproteins), and space-filling molecules [usually glycosaminoglycans (GAG), proteoglycans].
  • fibrous elements particularly collagen, elastin, or reticulin
  • cell adhesion polypeptides e.g., fibronectin, laminin and adhesive glycoproteins
  • space-filling molecules usually glycosaminoglycans (GAG), proteoglycans.
  • the decellularized ECM is derived from cardiac or pancreatic tissue.
  • tissues contemplated by the present inventors include brain tissue, bone tissue, muscle, liver, kidney, blood vessel, lung and placenta.
  • the decellularized ECM is not derived from fat tissue.
  • the decellularized ECM is not MATRIGEL or derived from isolated basement membranes.
  • the ECM may be derived from an autologous or non-autologous subject (e.g., from allogeneic or even xenogeneic tissue, due to non-immunogenicity of the resultant decellularized matrix).
  • PBS phosphate buffered saline
  • antibiotics such as Penicillin/Streptomycin 250 units/ml.
  • whole tissues can be used
  • the tissue may be washed at room temperature by agitation in large amounts (e.g., 50 ml per each gram of tissue segment) of EDTA solution (0.5-10 mM, pH-7.4).
  • the tissue is subjected to a hypertonic or hypotonic buffer to thereby obtain increased intercellular space within the tissue.
  • the hypertonic buffer used by the present invention can be any buffer or solution with a concentration of solutes that is higher than that present in the cytoplasm and/or the intercellular liquid within the tissue [e.g., a concentration of NaCl which is higher than 0.9% (w/v)]. Due to osmosis, incubation of the tissue with the hypertonic buffer results in increased intercellular space within the tissue.
  • peracetic acid is used to decellularize the tissue.
  • the hypertonic buffer used by the method according to this aspect of the present invention includes sodium chloride (NaCl) at a concentration which is higher than 0.9% (w/v), preferably, higher than 1% (w/v), preferably, in the range of 1-1.2% (w/v), e.g., 1.1% (w/v).
  • NaCl sodium chloride
  • the hypotonic buffer used by the method according to this aspect of the present invention includes sodium chloride (NaCl) at a concentration which is lower than 0.9% (w/v), lower than 0.8% (w/v), lower than 0.7% (w/v), preferably, in the range of 0.6-0.9 % (w/v), e.g., 0.7 % (w/v).
  • NaCl sodium chloride
  • the tissue is subjected to the hypertonic or hypotonic buffer for a time period leading to the biological effect, i.e., cell shrinkage which leads to increased intercellular space within the tissue.
  • the tissue is contacted with a hypertonic buffer (e.g. 1.1% w/v) and subsequently contacted with a hypotonic buffer (e.g. 0.7% w/v). This procedure may be repeated for two or more cycles.
  • a hypertonic buffer e.g. 1.1% w/v
  • a hypotonic buffer e.g. 0.7% w/v
  • the hypotonic buffer used by the method according to this aspect of the present invention includes sodium chloride (NaCl) at a concentration which is lower than 0.9% (w/v), lower than 0.8% (w/v), lower than 0.7% (w/v), preferably, in the range of 0.6-0.9% (w/v), e.g., 0.7% w/v.
  • NaCl sodium chloride
  • the tissue is further subjected to an enzymatic proteolytic digestion which digests all cellular components within the tissue yet preserves the ECM components (e.g., collagen and elastin) and thus results in a matrix which exhibits the mechanical and structural properties of the original tissue ECM.
  • ECM components e.g., collagen and elastin
  • measures are taken to preserve the ECM components while digesting the cellular components of the tissue. These measures are further described hereinbelow and include, for example, adjusting the concentration of the active ingredient (e.g., trypsin) within the digestion solution as well as the incubation time.
  • proteolytic digestion can be effected using a variety of proteolytic enzymes.
  • suitable proteolytic enzymes include trypsin and pancreatin which are available from various sources such as from Sigma (St Louis, Mo., USA).
  • proteolytic digestion is effected using trypsin.
  • Digestion with trypsin is preferably effected at a trypsin concentration ranging from 0.01-0.25% (w/v), more preferably, 0.02-0.2% (w/v), more preferably, 0.05-0.1 (w/v), even more preferably, a trypsin concentration of about 0.05% (w/v).
  • the concentration of the digestion solution and the incubation time therein depend on the type of tissue being treated and the size of tissue segments utilized and those of skilled in the art are capable of adjusting the conditions according to the desired size and type of tissue.
  • the tissue segments are incubated for at least about 20 hours, more preferably, at least about 24 hours.
  • the digestion solution is replaced at least once such that the overall incubation time in the digestion solution is at least 40-48 hours.
  • the detergent solution used by the method according to this aspect of the present invention includes TRITON-X-100 (available from Merck).
  • TRITON-X-100 is provided at a concentration range of 0.05-2.5% (v/v), more preferably, at 0.05-2% (v/v), more preferably at 0.1-2% (v/v), even more preferably at a concentration of 1% (v/v).
  • the detergent solution includes also ammonium hydroxide, which together with the TRITON-X-100, assists in breaking and dissolving cell nuclei, skeletal proteins, and membranes.
  • ammonium hydroxide is provided at a concentration of 0.05-1.5% (v/v), more preferably, at a concentration of 0.05-1% v/v), even more preferably, at a concentration of 0.1-1% (v/v) (e.g., 0.1%).
  • concentrations of TRITON-X-100 and ammonium hydroxide in the detergent solution may vary, depending on the type and size of tissue being treated and those of skills in the art are capable of adjusting such concentration according to the tissue used.
  • the above described detergent solution is preferably removed by subjecting the ECM to several washes in water or saline (e.g., at least 10 washes of 30 minutes each, and 2-3 washes of 24 hours each), until there is no evident of detergent solution in the matrix.
  • the decellularized ECM is then sterilized.
  • Sterilization of the decellularized ECM may be effected using methods known in the art (e.g. 70% ethanol).
  • the decellularized ECM typically, in order to carry out solubilization of the decellularized ECM, it is frozen (e.g. in liquid nitrogen), cut into small pieces (e.g. crumbled, crushed or ground) and then lyophilized.
  • the lyophilized decellularized ECM is then solubilized in an organic solvent.
  • organic solvents contemplated by the present inventors include, but are not limited to acetone, N,N-dimethylformamide (DMF), diethylformamide, chloroform, methylethylketone, acetic acid, formic acid, ethanol, 1,1,1,3,3,3-hexa fluoro-2-propanol (HTIP), tetrafluoroethanol, dichloromethane (DCM), tetrahydrofuran (THF), trifluoroacetic acid (TFA), camphorsulfonic acid, dimethyl acetamide, isopropyl alcohol (IPA) and mixtures thereof.
  • DMF 1,1,1,3,3,3-hexa fluoro-2-propanol
  • DCM dichloromethane
  • THF tetrahydrofuran
  • THF trifluoroacetic acid
  • camphorsulfonic acid dimethyl acetamide
  • Exemplary concentrations of decellularized ECM in the organic solvent contemplated by the present invention are between 0.005 g/mL to 0.5 g/mL—for example about 0.05 g/mL.
  • the organic solvent is HFIP.
  • the decellularized ECM may be homogenized.
  • the homogenization is effected in the presence of a rigid grinding media which is preferably spherical or particulate in form having an average size less than about 10 mm (e.g. between 2-10 mm) and, more preferably between 2-7 mm.
  • a rigid grinding media which is preferably spherical or particulate in form having an average size less than about 10 mm (e.g. between 2-10 mm) and, more preferably between 2-7 mm.
  • the selection of material for the grinding media is not believed to be critical.
  • Zirconium oxide, such as 95% ZrO stabilized with magnesia, zirconium silicate, ceramic, stainless steel, titania, alumina, 95% ZrO stabilized with yttrium, glass grinding media, and polymeric grinding media are exemplary grinding materials.
  • the grinding media can comprise particles that are preferably substantially spherical in shape, e.g., beads ; consisting essentially of polymeric resin or other suitable material.
  • the grinding media can comprise a core having a coating of a polymeric resin adhered thereon.
  • the homogenization may be performed using a homogenizer e.g, a bead homogenizer such as g a PrecellysTM 24 bead.
  • a homogenizer e.g, a bead homogenizer such as g a PrecellysTM 24 bead.
  • the homogenization should be effected for a length of time until the solution appears homogeneous (at 6000 rpm for 5 second intervals, for at least 6 intervals).
  • the homogenate may be sonicated.
  • decellularized ECM derived from the pancreas is sonicated (for example, for no more than three minutes) following the homogenization step.
  • the homogenate may be mixed for a suitable length of time (e.g. 1 day, two days, three days or more) by placing on a rotator.
  • a suitable length of time e.g. 1 day, two days, three days or more
  • the solution is then electrospun.
  • the polymer is a biocompatible polymer.
  • the polymer is a hydrophilic polymer.
  • the polymer is a synthetic polymer.
  • Exemplary synthetic polymers contemplated by the present invention include, but are not limited to poly(D,L-lactide) (PLA), poly(urethanes), poly(siloxanes), poly(silicones), poly(ethylene), polyvinyl pyrrolidone), poly(2-hydroxy ethyl methacrylate), poly(N-vinyl pyrrolidone), poly(methyl methacrylate), polyvinyl alcohol) (PVA), poly(acrylic acid), poly(vinyl acetate), polyacrylamide, poly(ethylene-co-vinyl acetate), poly(ethylene glycol), poly(methacrylic acid), polyglycolic acids (PGA), poly(lactide-co-glycolides) (PLEA), nylons, polyamides, polyanhydrides, poly(ethylene-co-vinyl alcohol) (EVOH), polycaprolactone, poly(vinyl acetate), polyvinylhydroxide, poly(ethylene oxide) (PEO), polyorthoesters and
  • the polymer is PEO.
  • the amount of polymer (e.g. PEO) in the solution is typically between 0.05-1% mass, and more preferably between 0.05-0.5% mass—for example about 0.1%.
  • electrospun fibers e.g. nanofibers and/or microfibers
  • electrospun fibers e.g. nanofibers and/or microfibers
  • An electrostatic field is employed to generate a positively charged jet from the dispenser to the collector.
  • a dispenser e.g., a syringe with metallic needle
  • a source of high voltage preferably of positive polarity
  • the collector is grounded, thus forming an electrostatic field between the dispenser and the collector.
  • the dispenser can be grounded while the collector is connected to a source of high voltage, preferably with negative polarity.
  • the fibers are electrospun with a voltage of 1-20 kV, for example between 5-10 kV and more preferably between 8-9 kV.
  • the hole of the dispenser may be between 20-30 gauge (e.g. a 23 gauge blunt needle), and the distance from needle to collector may be between 1-20 cm, more preferably between 6-10 cm, with a flow rate between 0.1-3 ml/hr, more preferably between 0.5-1 ml/hr.
  • the electrospun fibers are collected on a solid surface such as a metal surface or a polymeric surface.
  • the fibers are collected on a metal surface coated with a polymer (for example polyethylene).
  • a polymer is added to the dissolved decellularized ECM.
  • the present invention contemplates removing the polymer following the electrospinning, especially if the polymer is not biocompatible.
  • the polymer is a hydrophilic polymer, it may be removed simply by rinsing in an aqueous solution.
  • the scaffolds comprise electrospun decellularized ECM of an organ, wherein the decellularized ECM has a similar protein composition to native ECM of the organ.
  • scaffold refers to a three dimensional structure comprising a biocompatible material that provides a surface suitable for adherence and proliferation of cells.
  • a scaffold may further provide mechanical stability and support.
  • the dimensions and shape of the scaffold will vary according to the disease or injury being treated. It will be further appreciated that the dimensions of the scaffold will vary according to the size of the subject.
  • the scaffolds of the present invention are porous.
  • the porosity of the scaffold may be controlled by altering the parameters used for electrospinning, as known to those skilled in the art.
  • the minimum pore size and degree of porosity is dictated by the need to provide enough room for the cells and for nutrients to filter through the scaffold to the cells.
  • the maximum pore size and porosity is limited by the ability of the scaffold to maintain its mechanical stability after seeding.
  • the scaffold has an average pore diameter of about 10-40 ⁇ m.
  • the decellularized ECM may be derived from any tissue such as cardiac tissue, pancreatic tissue, blood vessel tissue, muscle tissue, liver tissue, kidney tissue, brain tissue, bone tissue, lung and placenta.
  • the decellularized ECM is derived from pancreatic tissue or cardiac tissue.
  • decellularized ECM When the decellularized ECM is derived from cardiac tissue, decellularized ECM maintains the protein composition of native cardiac ECM, or at least has a similar protein composition to native cardiac ECM.
  • the majority of the collagen of the decellularized ECM is collagen type I and type III, since that is the most abundant collagen in cardiac tissue.
  • the decellularized ECM is devoid of collagen type VI.
  • decellularized ECM When the decellularized ECM is derived from pancreatic tissue, decellularized ECM maintains the protein composition of native pancreatic ECM or at least has a similar protein composition to native pancreatic ECM.
  • the majority of the collagen of the decellularized ECM are collagens type I and type III, since that is the most abundant collagen in pancreatic tissue.
  • the scaffold comprises electrospun decellularized ECM, wherein the decellularized ECM is derived from an organ selected from the group consisting of heart and pancreas.
  • the scaffold comprises fibers of different thickness.
  • thick fibers may have an average diameter between 100-2000 nm, more preferably between 300-1500 nm (which corresponds to type I collagen fibers in native ECM) and thin fibers have an average diameter of between 30-80 nm (which corresponds to type III collagen fibers in native ECM).
  • scaffolds generated according to methods described herein are of a similar protein composition to native ECM, have a similar fiber diameter to native ECM and/or have a similar organization to native ECM when hydrated.
  • the scaffolds are devoid of a synthetic polymer (do not comprise more than trace amounts of synthetic polymer).
  • Therapeutic compounds or agents that modify cellular activity can also be incorporated (e.g. attached to, coated on, embedded or impregnated) into the scaffold material. Furthermore, the present inventors contemplate embedding particles which release the therapeutic compounds or agents into the scaffold.
  • Campbell et al US Patent Application No. 20030125410 which is incorporated by reference as if fully set forth by reference herein, discloses methods for fabrication of 3D scaffolds for stem cell growth, the scaffolds having preformed gradients of therapeutic compounds.
  • the scaffold materials, according to Campbell et at, fall within the category of “bio-inks”, Such “bio-inks” are suitable for use with the compositions and methods of the present invention.
  • agents that may be incorporated into the scaffold of the present invention include, but are not limited to those that promote cell adhesion (e.g. fibronectin, integrins), cell colonization, cell proliferation, cell differentiation, anti-inflammatories, cell extravasation and/or cell migration.
  • the agent may be an amino acid, a small molecule chemical, a peptide, a polypeptide, a protein, a DNA, an RNA, a lipid and/or a proteoglycan.
  • Proteins that may be incorporated into the scaffolds of the present invention include, but are not limited to extracellular matrix proteins, cell adhesion proteins, growth factors, cytokines, hormones, proteases and protease substrates.
  • exemplary proteins include vascular endothelial-derived growth factor (Vain, activin-A, retinoic acid, epidermal growth factor, bone morphogenetic protein, TGF ⁇ , hepatocyte growth factor, platelet-derived growth factor, TGF ⁇ , IGF-I and II, hematopoetic growth factors, heparin binding growth factor, peptide growth factors, erythropoietin, interleukins, tumor necrosis factors, interferons, colony stimulating factors, basic and acidic fibroblast growth factors, nerve growth factor (NGF) or muscle morphogenic factor (MMP).
  • NGF nerve growth factor
  • MMP muscle morphogenic factor
  • the particular growth factor employed should be appropriate to the desired cell activity.
  • the scaffolds of the invention may be seeded with cells, including for example primary cells, cultured cells, single cell suspensions of cells, clusters of cells e.g. islets, cells which are comprised in tissues and/or organs etc.
  • cells including for example primary cells, cultured cells, single cell suspensions of cells, clusters of cells e.g. islets, cells which are comprised in tissues and/or organs etc.
  • Cells can be seeded in the scaffold by static loading, or, more preferably, by seeding in stirred flask bioreactors (scaffold is typically suspended from a solid support), in a rotating wall vessel, or using direct perfusion of the cells in medium in a bioreactor. Highest cell density throughout the scaffold is achieved by the latter (direct perfusion) technique.
  • the cells may be seeded directly onto the scaffold, or alternatively, the cells may be mixed with a gel which is then absorbed onto the interior and exterior surfaces of the scaffold and which may fill some of the pores of the scaffold. Capillary forces will retain the gel on the scaffold before hardening, or the gel may be allowed to harden on the scaffold to become more self-supporting.
  • the cells may be combined with a cell support substrate in the form of a gel optionally including extracellular matrix components.
  • An exemplary gel is MatrigelTM, from Becton-Dickinson. MatrigelTM is a solubilized basement membrane matrix extracted from the EHS mouse tumor (Kleinman, H. K., et al, Biochem. 25:312, 1986).
  • the primary components of the matrix are laminin, collagen I, entactin, and heparan sulfate proteoglycan (perlecan) (Vukicevic, S., et al., Exp. Cell Res. 202:1, 1992).
  • MatrigelTM also contains growth factors, matrix metalloproteinases (MMPs [collagenases]), and other proteinases (plasminogen activators [PAs]) (Mackay, A. R., et al., BioTechniques 15:1048, 1993).
  • MMPs [collagenases] matrix metalloproteinases
  • PAs proteinases
  • the matrix also includes several undefined compounds (Kleinman, H. K., et al., Biochem. 25:312, 1986; McGuire, P. G. and Seeds, N. W., J. Cell. Biochem. 40:215, 1989), but it does not contain any detectable levels of tissue inhibitors of metalloproteinases (TIMPs) (Mac
  • the gel may be growth-factor reduced Matrigel, produced by removing most of the growth factors from the gel (see Taub, et at., Proc. Natl. Acad. Sci. USA (1990); 87 (10:4002-6).
  • the gel may be a collagen I gel, alginate or agar.
  • Such a gel may also include other extracellular matrix components, such as glycosaminoglycans, fibrin, fibronectin, proteoglycans, and glycoproteins.
  • the gel may also include basement membrane components such as collagen IV and laminin.
  • Enzymes such as proteinases and collagenases may be added to the gel, as may cell response modifiers such as growth factors and chemotactic agents.
  • the cells may be derived from any organism including for example mammalian cells, (e.g. human), plant cells, insect cells, algae cells, fungal cells (e.g. yeast cells), prokaryotic cells (e.g. bacterial cells).
  • mammalian cells e.g. human
  • plant cells insect cells, algae cells, fungal cells (e.g. yeast cells), prokaryotic cells (e.g. bacterial cells).
  • the cells comprise stem cells—e.g. adult stem cells such as mesenchymal stem cells or pluripotent stem cells such as embryonic stem cells or induced pluripotent stem cells.
  • stem cells may be modified so as to undergo ex vivo differentiation.
  • the cells are preferably intact (i.e. whole), and preferably viable, although it will be appreciated that pre-treatment of cells, such as generation of cell extracts or non-intact cells are also contemplated by the present invention.
  • the cells may be fresh, frozen or preserved in any other way known in the art cryopreserved).
  • the cells are derived from the pancreas or the heart.
  • the tissue from which the decellularized extracellular matrix is produced may be selected (i.e. matched) according to the cells which are incorporated therein.
  • the tissue from which the decellularized extracellular matrix is produced is pancreatic tissue.
  • the cells are derived from cardiac tissue—e.g. cardiac myocardial cells (or modified so as to imitate cardiac myocardial cells), according to certain embodiments, the tissue from which the decellularized extracellular matrix is produced is cardiac myocardial tissue.
  • cardiac myocardial cells e.g. cardiac myocardial cells (or modified so as to imitate cardiac myocardial cells)
  • the tissue from which the decellularized extracellular matrix is produced is cardiac myocardial tissue.
  • the cells secrete a factor (e.g. a polypeptide) that is useful for the treatment of a disease.
  • a factor e.g. a polypeptide
  • hormones including but not limited to insulin, thyroxine, growth hormone, testosterone, oestrogen, erythropoietin and aldosterone
  • enzymes including but not limited to lysosomal enzyme such as glucocerebrosidase (GCD), acid sphingomyelinase, hexosaminidase, ⁇ -N-acetylgalactosaminidi se, acid lipase, ⁇ -galactosidase, ⁇ -L-iduronidase, iduronate sulfatase, ⁇ -mannosidase, sialidase, ⁇ fucosidase, G M1 - ⁇ -galctosidase, ceramide lactosidase, arylsulfatase A, ⁇ galactosidase and ceramidase; clotting factors such as factor VIII.
  • GCD glucocerebrosidase
  • the cells secrete insulin.
  • insulin refers to an insulin obtained by synthesis or recombination, in which the peptide sequence is the sequence of human insulin, includes the allelic variations and the homologs.
  • the polypeptide sequence of the insulin may be modified to improve the function of the insulin (e.g. long lasting).
  • the cells are na ⁇ ve (non-genetically modified).
  • the present invention also contemplates use of cells which have been genetically modified to express a recombinant protein.
  • the recombinant protein may be a therapeutic protein or may promote in vivo longevity (AM, adrenomedullin, Jun-Ichiro et al. Tissue Eng. 2006) or may promote neurotransmitter release (e.g., such as by transfecting with tyrosine hydroxylase).
  • therapeutic, recombinant proteins that may be expressed in the cells of the present invention include, but are not limited to an antibody, insulin, human growth hormone (rHGH), follicle stimulating hormone, factor VIII, erythropoietin, Granulocyte colony-stimulating factor (G-CSF),alpha-glactosidase A, alpha-L-iduronidase (rhIDU; laronidase),N-acetylgalactosamine-4-sulfatase (rhASB; galsulfase) Tissue plasminogen activator (TPA), Glucocerebrosidase, Interferon (IF) Interferon-beta-1a, Interferon beta-1b, Insulin-like growth factor 1 (IGF-1), somatotropin (ST) and chymosin.
  • rHGH human growth hormone
  • follicle stimulating hormone factor VIII
  • factor VIII erythropoietin
  • G-CSF Granulocyte
  • exogenous polynucleotides which may be expressed in accordance with the present teachings include, but are not limited to, polypeptides such as peptide hormones, antibodies or antibody fragments (e.g., Fab), enzymes and structural proteins or dsRNA, antisense/ribozyme transcripts which can be directed at specific target sequences transcripts of tumor associated genes) to thereby downregulate activity thereof and exert a therapeutic effect.
  • polypeptides such as peptide hormones, antibodies or antibody fragments (e.g., Fab)
  • enzymes and structural proteins or dsRNA e.g., antisense/ribozyme transcripts which can be directed at specific target sequences transcripts of tumor associated genes
  • antisense/ribozyme transcripts which can be directed at specific target sequences transcripts of tumor associated genes
  • protective protein antigens for vaccination see, for example, Babiuk S et al J Control Release 2000;66:199-214
  • enzymes such as fibrinolysin for treatment of
  • a method of treating a medical condition e.g., pathology, disease, syndrome, trauma
  • a medical condition e.g., pathology, disease, syndrome, trauma
  • transplanting the scaffold of the present invention into the subject comprising transplanting the scaffold of the present invention into the subject.
  • treating refers to inhibiting or arresting the development of a pathology and/or causing the reduction, remission, or regression of a pathology.
  • Those of skill in the art will understand that various methodologies and assays can be used to assess the development of a pathology, and similarly, various methodologies and assays may be used to assess the reduction, remission or regression of a pathology.
  • the term “treating” refers to alleviating or diminishing a symptom associated with a disease or trauma which may benefit from cell transplantation.
  • treating cures, e.g., substantially eliminates, the symptoms associated with the medical condition.
  • a medical condition which may benefit from cell transplantation refers to any medical condition which may be alleviated by administration of the scaffold (cell-seeded, or non cell-seeded) of the present invention.
  • Examples of such medical conditions include, but are not limited to, stem cell deficiency, heart disease, neurodegenerative diseases, glaucoma neuropathy, Parkinson's disease, cancer, Schizophrenia, Alzheimer's disease, stroke, burns, loss of tissue, loss of blood, anemia, autoimmune disorders, diabetes, arthritis, graft vs.
  • GvHD host disease
  • EAE autoimmune encephalomyelitis
  • SLE systemic lupus erythematosus
  • rheumatoid arthritis systemic sclerosis
  • MS multiple sclerosis
  • MG Myasthenia Gravis
  • GBS Guillain-Barre Syndrome
  • HT Hashimoto's Thyroiditis
  • IDM Insulin Dependent Diabetes Melitus
  • the method may be applied to repair cardiac tissue in a human subject having a cardiac disorder so as to thereby treat the disorder.
  • the method can also be applied to repair cardiac tissue susceptible to be associated with future onset or development of a cardiac disorder so as to thereby inhibit such onset or development.
  • the present invention can be advantageously used to treat disorders associated with, for example, necrotic, apoptotic, damaged, dysfunctional or morphologically abnormal myocardium.
  • disorders include, but are not limited to, ischemic heart disease, cardiac infarction, rheumatic heart disease, endocarditis, autoimmune cardiac disease, valvular heart disease, congenital heart disorders, cardiac rhythm disorders, impaired myocardial conductivity and cardiac insufficiency.
  • the method according to this aspect of the present invention can be advantageously used to efficiently reverse, inhibit or prevent cardiac damage caused by ischemia resulting from myocardial infarction.
  • the method according to this aspect of the present invention can be used to treat cardiac disorders characterized by abnormal cardiac rhythm, such as, for example, cardiac arrhythmia.
  • the method according to this aspect of the present invention can be used to treat impaired cardiac function resulting from tissue loss or dysfunction that occur at critical sites in the electrical conduction system of the heart, that may lead to inefficient rhythm initiation or impulse conduction resulting in abnormalities in heart rate.
  • a compound or “at least one compound” may include a plurality of compounds, including mixtures thereof.
  • a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range.
  • the phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.
  • Decellularization procedure Porcine cardiac or pancreas ECM was decellularized and sterilized according to a previously published protocol. 21 (Chaimov 2016, PMID: 27476611) Briefly, tissue of healthy commercial slaughter-weight pigs was isolated for the decellularization procedure. The procedure was comprised of two cycles with the following stages: Alternating hyper/hypo tonic NaCl solutions; enzymatic treatment using trypsin; and detergent washes with Triton-X-100.
  • PEO was added only to solutions of porcine cardiac (pcECM) to obtain a final solution of 0.1 mass % PEO.
  • the porcine pancreatic ECM (ppECM) or pcECM/PEO solution was electrospun using a custom built electrospinning device.
  • the fibers were electrospun with a voltage of 8-9 kV, and collected on a thin film of polyethylene covering a rotating aluminum disc with a diameter of 9 cm and a width of 11 mm.
  • the capillary was a 23 gauge blunt needle, and the distance from needle to collector was 6-10 cm, and the flow rate was 0.5-1 ml/hr. Fibers were collected until a thick matte was observed. The matte was then peeled off the surface and placed in a dry environment.
  • PEO was removed from pcECM matrices by washing in a water-based solution, while the pcECM fibers remained intact.
  • the contact angle of the ECM electrospun fibrous scaffolds was determined by disposing a droplet of near 1 mm in diameter onto the surface. A static image was then captured with an Artcam 130 MIBW camera (Artray Co. Ltd., Tokyo, Japan). The contact angle was then calculated using a special procedure developed in MatLab R2014a software (Mathworks, Natick, Mass., USA).
  • FTIR Fourier transform infrared spectroscopy
  • TGA Thermal gravimetric analysis
  • Electrospun ECM scaffold composition An analysis of the protein composition was performed at the Proteomics Center, Technion—Israel Institute of Technology. Samples were digested by trypsin and the resulting peptides were analyzed by LC-MS/MS. After which, the peptide mix was fractionated by HPLC and electro-sprayed onto an ion-trap mass spectrometer, in order to determine the proteins' mass. The peptides were further fragmented by collision induced dissociation and analyzed again, for additional analysis and identification. Peptides were analyzed and identified using Proteome DiscovererTM software (Thermo-Scientific) against the porcine part of the UniProt database.
  • hMSCs Human bone marrow mesenchymal stem cells (hMSCs, Lonza, Basel, Switzerland) were seeded (10,000 per scaffold) and cultured for 1 month. hMSCs were cultured in ⁇ MEM, supplemented with FCS (10%), pen-strep (1%), fimgizone (0.4%), and basic fibroblast growth factor (5 ng mL ⁇ 1 ). The medium was replaced every second day. Cell viability was evaluated using the AlamarBlueTM reagent (AbD Serotec, Kidlington, UK), according to the manufacturer's protocol. hiPSCs were seeded (30,000 per scaffold) and cultured for 1 month.
  • the hiPSCs were cultured in mTeSRTM Basal Medium and supplemented with mTeSRTM 1 5 ⁇ supplement. The medium was replaced every day.
  • Neonatal cardiomyocytes were isolated from 24-hr-old Wister rats. Excised hearts were minced, and the cardiac cells were dissociated by gentle agitation in 200U mL ⁇ 1 RDB in PBS-G (PBS, pen-strep, 0.1% D-glucose) at 37° c for 10 min. Cell suspensions were centrifuged at 1000 rpm for 5 min, suspended in F-10 nutrient mixture supplemented with 5% fetal bovine serum, 5% DHS, 1% pen-strep and 0.4% fungizone and 1 mM CaCl 2 .
  • LSFM Lightsheet Fluorescent Microscopy
  • Ca 2+ imaging Cells were loaded with 5 mM of fluo-4 fluorescent Ca 2+ indicator (Molecular Probes) in the presence of Plutonic F-127 (Molecular Probes) at a dilution of 2:1 to allow the recording of intracellular Ca 2+ -transients (whole-cell [Ca 2+ ] transients).
  • scaffolds were plated on a 35-mm optical plate (Matek) with field simulation electrodes (RC-37FS; Warner Instruments), and paced using a stimulus isolation unit (SIU-102, Warner Instruments), by applying 5 ms-suprathreshold bipolar stimulation pulses up to 50 mA.
  • Intracellular Ca 2+ -transients were recorded using a Zeiss laser-scanning confocal imaging system (Flux-view; Olympus) mounted on an upright BX151WI Olympus microscope equipped with a X60 water objective. Data were analyzed utilizing MatLab-based custom-written software.
  • ECM was obtained from slices of porcine tissues that were decellularized as described in WO 2006095342A2 and in the Materials and methods section herein above, which avoided the use of SDS and allowed for improved preservation of ECM biological activity.
  • ECM were frozen in liquid nitrogen, ground in a cryogenic tissue grinder, and lyophilized. The resulting powder was used for dissolution.
  • ECM electrospun fibrous scaffolds composition Since the protein composition is a major factor contributing to the biological activity and mechanical properties of the scaffold, possible changes in protein composition were first evaluated by comparing FTIR spectra obtained from decellularized pcECM to those of the ES-pcECM scaffold ( FIG. 9A-B ). Both materials exhibited amide vibrations, near and above 3000 cm ⁇ 1 , characteristic of peptide groups, and vibrations between 500-1700 cm ⁇ 1 , characteristic of amino acid side chains, with no significant differences between decellularized pcECM and ES-pcECM scaffold. Moreover, similar percentages of each secondary structure were determined by Fourier deconvolution.
  • Table 2 presents the collagenous composition of electrospun porcine pancreatic ECM (ppECM) scaffold as revealed in proteomic analysis.
  • Levels 2 and 3 contain the less abundant fibrillar forming collagens (II and V), and network forming collagen that is present in basement membranes (IV). Level 3 also contains collagen type VI, which provides a microfilament network that organizes the fibrillary collagens and anchors them to the basement membranes.
  • Collagen alpha-2 (VI) was the only collagen that exhibited a significant fold decrease in the pcECM electrospun scaffold from the decellularized pcECM. In general, it was determined that, with the exception of collagen alpha-2 (VI), there was no significant fold difference in the collagen content between the decellularized pcECM and the pcECM electrospun scaffold,
  • TGA analyses ( FIG. 10 ), compared the characteristic thermal degradation of the scaffold to that of decellularized pcECM.
  • the onset of decomposition (T onset ) was similar for both materials: 259.4° C. (scaffold) and 259.6° C. (decellularized pcECM), signifying that the production process of ES-pcECM scaffolds, and particularly the dissolution of pcECM in HFIP, had not degraded the collagen.
  • Self assembly of electrospun peECM Upon wetting the ES-pcECM scaffold, the electrospun fibers underwent self-assembly, consequently obviating the need for a synthetic cross-linking agent ( FIG. 11 ).
  • the extent of self-assembly varied according to the initial wetting temperature as well as the subsequent incubation temperature. The largest degree of self-assembly was obtained when ES-pcECM scaffolds were maintained at 37° C., where the ES-pcECM self-assembly produced an isotropic, porous, ordered structure that most clearly and uniquely resembled the microstructure of native pcECM.
  • ES-pcECM scaffolds maintained at 24° C.
  • Mechanical properties of the electrospun pcECM scaffold The mechanical properties of an engineered scaffold are particularly critical when addressing cardiac regeneration.
  • the mechanical properties of the electrospun pcECM scaffold were compared to those of native tissue in a physiological solution ( FIG. 12 A-D).
  • the stress and strain at maximum stress were similar in the electrospun pcECM scaffold and native cardiac tissue (p>0.05).
  • the Young's modulus (E) of the scaffolds (029 ⁇ 0.007 MPa) was also comparable to that of native tissue (0.22 ⁇ 0.007), though statistically to different (p ⁇ 0.05).
  • hMSCs human mesenchymal stem cells
  • FIGS. 14A-D hMSCs portrayed normal elongated morphology and scaffold penetration.
  • FIGS. 14A-D The presence of adherent, viable hMSCs after 1 month of culture on the scaffold was also supported by light sheet fluorescence microscopy (LSFM) analysis ( FIGS. 15A-B ).
  • LSFM light sheet fluorescence microscopy
  • FIGS. 15A-B Hematoxylin and eosin (II&E) histological analysis demonstrated that the cells had integrated within the matrix ( FIG. 15C ).
  • the seeded hMSCs' ability to remodel the electrospun pcECM scaffold was evaluated by analyzing their expression of ECM-remodeling related genes ( FIG. 16 ).
  • collagen I expression increased 3, 7, and 14 days post seeding in 10-20 folds compare to the basal level. This elevated expression decreased by day 21.
  • Maximal increase of collagen III expression was observed in day 3 for the native ECM (13 fold) and day 21 for the electrospun ECM scaffold (18 fold).
  • TIMP1 collagenase inhibitor
  • maximal expression was observed at day 7 for the electrospun scaffold (25 fold) and day 14 for the native ECM (7 fold).
  • MMP2 an indicator collagenase, increased at day 7, and reached maximal expression at day 14 on both native ECM and the electrospun ECM scaffold.
  • hiPSC Human induced pluripotent stem cells
  • Neonatal rat cardiomyocytes (rCM) seeded on the electrospun pcECM scaffolds were assessed after 21 days using SEM and LSFM ( FIGS. 19A-B ), and revealed normal spherical morphology. H&E staining demonstrated the cells' integration within the matrix ( FIG. 19C ).
  • the seeded rCM were positively stained for the typical functional cardiac proteins cardiac troponin I (cTn1), sarcomeric alpha-actinin and connexin-43 ( FIG. 20A-C ), indicating contractile functioning as well as cell—cell coupling through functional gap junctions. This contractile function was also demonstrated through the spontaneous beating of the seeded scaffolds, initiated less than 2 days post seeding.
  • the synchronically beating scaffold was analyzed using Ca 2+ imaging to record beating and to test electrical coupling.
  • the action potential properties evaluated during pacing (using field stimuli) at different rates ( FIG. 21 ) further confirmed that the electrospun pcECM scaffold can support the cells' intact synchronized electrical activity.
  • hiPSC-derived cardiomyocytes hiPSC-derived cardiomyocytes (hiPSC-CM)—on electrospun pcECM scaffold revealed high viability levels 14 days and 3 weeks post seeding ( FIGS. 22 , FIG. 23 ). Contractile function was also demonstrated through the spontaneous beating of the seeded scaffolds, initiated less than 24 hr post seeding. After approximately two weeks, the synchronically beating scaffolds was analyzed using Ca 2+ imaging to test electrical coupling. As seen in FIG.
  • the expression of the pro-inflammatory cytokine TNF- ⁇ showed a 1.66 ⁇ 0.64 fold increase for RAW macrophages stimulated with pcECM scaffolds, while there exhibited at 0.73 ⁇ 0.65- and a 67.5 ⁇ 32.6-fold increase for RAW macrophages stimulated with PLGA and LPS, respectively it1 respect to non-stimulated cells). Only the fold increase of the TNF- ⁇ expression exhibited by RAW macrophages stimulated with LPS can be considered highly significant (P ⁇ 0.01).
  • the expression of the pro-inflammatory cytokine IL1- ⁇ showed a 2.69 ⁇ 1.79 fold increase for RAW macrophages stimulated with pcECM scaffolds, while there exhibited at 0.25 ⁇ 0.26-fold and a 18,124 ⁇ 14,581-fold increase for RAW macrophages stimulated with PLGA and LPS, respectively (with respect to non-stimulated cells). Only the fold increase of the IL1- ⁇ expression exhibited by RAW macrophages stimulated with LPS can be considered highly significant (P ⁇ 0.01).
  • the immunogenicity of pcECM electrospun fibrous scaffolds was additionally evaluated in vivo through subcutaneous implantation. Mice were split into two groups; one group received the pcECM scaffold, and the other received electrospun PLGA scaffolds as a negative control. One, two and four weeks following implantation the mice were sacrificed. Lymph node examination revealed no swelling or irritation in all tested groups. In addition, the expression of pro-inflammatory cytokines TNF- ⁇ and IL1- ⁇ in the lymph nodes revealed no significant difference between the ES-pcECM scaffold groups and the ES-PLGA scaffold groups of the same week ( FIGS. 26A-B ).
  • CBC Complete blood counts revealed no increase in the levels of white blood cells (WBCs), red blood cells (RBCs), hematocrit, hemoglobin, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), MCH concentration (MCHC), neutrophils, and lymphocytes at the ES-pcECM scaffold treatment group compared to the PLGA negative control group for all time points ( FIG. 27 A-I).
  • Kitsara M. et al. Fabrication of cardiac patch by using electrospun collagen fibers. Microelectronic Engineering 144, 46-50 (2015).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Biomedical Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Epidemiology (AREA)
  • Dermatology (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Transplantation (AREA)
  • Cell Biology (AREA)
  • Zoology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Diabetes (AREA)
  • Botany (AREA)
  • Developmental Biology & Embryology (AREA)
  • Hematology (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Biophysics (AREA)
  • Urology & Nephrology (AREA)
  • Molecular Biology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Cardiology (AREA)
  • Biotechnology (AREA)
  • Virology (AREA)
  • Emergency Medicine (AREA)
  • Endocrinology (AREA)
  • Obesity (AREA)
  • Materials For Medical Uses (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
US16/092,787 2016-04-12 2017-04-06 Scaffolds fabricated from electrospun decellularized extracellular matrix Abandoned US20190142998A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
IL245059 2016-04-12
IL245059A IL245059A0 (en) 2016-04-12 2016-04-12 Electrospun scaffolds from extracellular matrix
PCT/IL2017/050428 WO2017179042A1 (en) 2016-04-12 2017-04-06 Scaffolds fabricated from electrospun decellularized extracellular matrix

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/IL2017/050428 A-371-Of-International WO2017179042A1 (en) 2016-04-12 2017-04-06 Scaffolds fabricated from electrospun decellularized extracellular matrix

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US18/100,000 Division US20230158208A1 (en) 2016-04-12 2023-01-23 Scaffolds fabricated from electrospun decellularized extracellular matrix

Publications (1)

Publication Number Publication Date
US20190142998A1 true US20190142998A1 (en) 2019-05-16

Family

ID=56241717

Family Applications (2)

Application Number Title Priority Date Filing Date
US16/092,787 Abandoned US20190142998A1 (en) 2016-04-12 2017-04-06 Scaffolds fabricated from electrospun decellularized extracellular matrix
US18/100,000 Pending US20230158208A1 (en) 2016-04-12 2023-01-23 Scaffolds fabricated from electrospun decellularized extracellular matrix

Family Applications After (1)

Application Number Title Priority Date Filing Date
US18/100,000 Pending US20230158208A1 (en) 2016-04-12 2023-01-23 Scaffolds fabricated from electrospun decellularized extracellular matrix

Country Status (5)

Country Link
US (2) US20190142998A1 (zh)
EP (1) EP3442563A4 (zh)
CN (1) CN109475606A (zh)
IL (2) IL245059A0 (zh)
WO (1) WO2017179042A1 (zh)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111603609A (zh) * 2020-05-25 2020-09-01 医工瑞思(福建)工程研究中心有限公司 一种仿生组织工程支架及其制备方法
WO2022050393A1 (ja) * 2020-09-03 2022-03-10 凸版印刷株式会社 三次元組織体の製造方法及び三次元組織体
CN116747355A (zh) * 2023-07-26 2023-09-15 鑫华微(厦门)生物科技有限公司 一种复合人工皮肤及其制备方法

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11904069B2 (en) * 2017-04-04 2024-02-20 University Of Pittsburgh-Of The Commonwealth System Of Higher Education Mg alloy mesh reinforced polymer/ECM hybrid scaffolds for critical-sized bone defect regeneration
CN108642576A (zh) * 2018-05-16 2018-10-12 中山大学 一种细胞外基质的静电纺丝方法及静电纺丝纤维
CN110464880B (zh) * 2019-09-27 2021-10-26 中国医学科学院生物医学工程研究所 一种人工胰岛或人工胰腺及其制备方法
CN111544651A (zh) * 2020-05-19 2020-08-18 南通大学附属医院 基于静电纺丝的丝素蛋白/胰腺脱细胞基质混合支架
CN115453021B (zh) * 2022-08-15 2023-11-14 首都医科大学附属北京友谊医院 一种sds脱细胞溶液及其在脱细胞处理或细胞外基质不可溶组分鉴定中的应用
CN117205373B (zh) * 2023-11-08 2024-02-02 独步吾奇生物医疗科技(江苏)有限公司 一种胶原支架及其制备方法和应用

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8932620B2 (en) * 2005-06-17 2015-01-13 Drexel University Three-dimensional scaffolds for tissue engineering made by processing complex extracts of natural extracellular matrices
EP2349292B1 (en) * 2008-09-30 2017-04-12 The Regents of the University of California Compositions comprising decellularized extracellular matrix obtained from cardiac tissue
ES2758782T3 (es) * 2010-06-01 2020-05-06 Dsm Ip Assets Bv Extracción de lípido de células y productos del mismo
US9421307B2 (en) * 2010-08-17 2016-08-23 University of Pittsburgh—of the Commonwealth System of Higher Education Biohybrid composite scaffold
CA2863967C (en) * 2012-02-07 2021-02-09 Lifenet Health Liquefaction of bone matrix

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111603609A (zh) * 2020-05-25 2020-09-01 医工瑞思(福建)工程研究中心有限公司 一种仿生组织工程支架及其制备方法
WO2022050393A1 (ja) * 2020-09-03 2022-03-10 凸版印刷株式会社 三次元組織体の製造方法及び三次元組織体
CN116747355A (zh) * 2023-07-26 2023-09-15 鑫华微(厦门)生物科技有限公司 一种复合人工皮肤及其制备方法

Also Published As

Publication number Publication date
IL262318B1 (en) 2024-05-01
IL262318A (en) 2018-11-29
EP3442563A1 (en) 2019-02-20
EP3442563A4 (en) 2020-01-08
IL245059A0 (en) 2016-06-30
CN109475606A (zh) 2019-03-15
US20230158208A1 (en) 2023-05-25
WO2017179042A1 (en) 2017-10-19

Similar Documents

Publication Publication Date Title
US20230158208A1 (en) Scaffolds fabricated from electrospun decellularized extracellular matrix
Schoen et al. Electrospun extracellular matrix: Paving the way to tailor‐made natural scaffolds for cardiac tissue regeneration
Zhang et al. Photo-crosslinkable amniotic membrane hydrogel for skin defect healing
US10220116B2 (en) Liquefaction of bone matrix
Wang et al. Evaluation of the potential of rhTGF-β3 encapsulated P (LLA-CL)/collagen nanofibers for tracheal cartilage regeneration using mesenchymal stems cells derived from Wharton's jelly of human umbilical cord
Lih et al. Biomimetic porous PLGA scaffolds incorporating decellularized extracellular matrix for kidney tissue regeneration
US20200276360A1 (en) Compositions and methods for cardiac therapy
Yang et al. The cardiomyogenic differentiation of rat mesenchymal stem cells on silk fibroin–polysaccharide cardiac patches in vitro
EP2254608B1 (en) Compartmental extract compositions for tissue engineering
Del Gaudio et al. Induction of angiogenesis using VEGF releasing genipin-crosslinked electrospun gelatin mats
Ravichandran et al. Poly (glycerol sebacate)/gelatin core/shell fibrous structure for regeneration of myocardial infarction
CN110169959B (zh) 生长因子缓释微球、组织工程软骨复合支架及制备方法
Yeo et al. Photocrosslinkable hydrogel for myocyte cell culture and injection
US9216236B2 (en) Natural tissue-derived decellularized matrix and methods of generating and using same
Biazar et al. The healing effect of stem cells loaded in nanofibrous scaffolds on full thickness skin defects
US20060153815A1 (en) Tissue engineering devices for the repair and regeneration of tissue
WO2017136786A1 (en) Rigionally specific tissua-derived extracellular metrix
Kai et al. Potential of VEGF‐encapsulated electrospun nanofibers for in vitro cardiomyogenic differentiation of human mesenchymal stem cells
EP2999486B1 (en) Metal-coated scaffolds for tissue engineering
Rajabi et al. Effect of chemical immobilization of SDF‐1α into muscle‐derived scaffolds on angiogenesis and muscle progenitor recruitment
Kong et al. Nerve decellularized matrix composite scaffold with high antibacterial activity for nerve regeneration
Liu et al. Decellularized extracellular matrix materials for treatment of ischemic cardiomyopathy
Li et al. L-polylactic acid porous microspheres enhance the mechanical properties and in vivo stability of degummed silk/silk fibroin/gelatin scaffold
JP2023501925A (ja) 筋線維断片を利用した回旋腱板療法
Jiang et al. SDF-1α and CTGF functionalized Gelatin methacryloyl (GelMA) hydrogels enhance fibroblast activation to promote wound healing

Legal Events

Date Code Title Description
AS Assignment

Owner name: TECHNION RESEARCH & DEVELOPMENT FOUNDATION LIMITED

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:MACHLUF, MARCELLE;ZUSSMAN, EYAL;BARUCH, LIMOR;SIGNING DATES FROM 20181014 TO 20181015;REEL/FRAME:048555/0507

STPP Information on status: patent application and granting procedure in general

Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION

STPP Information on status: patent application and granting procedure in general

Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER

STPP Information on status: patent application and granting procedure in general

Free format text: NON FINAL ACTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER

STPP Information on status: patent application and granting procedure in general

Free format text: FINAL REJECTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: RESPONSE AFTER FINAL ACTION FORWARDED TO EXAMINER

STPP Information on status: patent application and granting procedure in general

Free format text: ADVISORY ACTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION

STPP Information on status: patent application and granting procedure in general

Free format text: NON FINAL ACTION MAILED

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION