WO2014036094A1 - Isolation de fraction vasculaire stromale des tissus adipeux, obtenue à l'aide de l'homogénéisation avec des perles - Google Patents

Isolation de fraction vasculaire stromale des tissus adipeux, obtenue à l'aide de l'homogénéisation avec des perles Download PDF

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WO2014036094A1
WO2014036094A1 PCT/US2013/057007 US2013057007W WO2014036094A1 WO 2014036094 A1 WO2014036094 A1 WO 2014036094A1 US 2013057007 W US2013057007 W US 2013057007W WO 2014036094 A1 WO2014036094 A1 WO 2014036094A1
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cells
stromal vascular
vascular fraction
adipose tissue
beads
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PCT/US2013/057007
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English (en)
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Steven VICTOR
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Intellicell Biosciences Inc.
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    • 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
    • A61K35/35—Fat tissue; Adipocytes; Stromal cells; Connective tissues

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  • the present invention relates to the isolation of a stromal vascular fraction from adipose tissue comprising subjecting adipose tissue to homogenization with beads, wherein the adipose cells and blood vessels are lysed thereby releasing intact stromal vascular fraction cells while substantially maintaining the viability of the stromal vascular fraction.
  • Adipose tissue comprises two fractions— the adipocyte fraction and the stromal-vascular fraction (SVF).
  • Adipose tissue may be found in the visceral adipose tissue (VAT) and subcutaneous adipose tissue deposits (SAT).
  • VAT visceral adipose tissue
  • SAT subcutaneous adipose tissue deposits
  • the stromal vascular fraction (SVF) comprises preadipocytes, mesenchymal stem cells (MSC), endothelial progenitor cells, blood endothelial cells, fibroblasts, pericytes, T regulatory cells, and macrophages.
  • MSC mesenchymal stem cells
  • the mesenchymanl stem cells (MSC) can be differentiated into a variety of cell lineages including adipogenic, chondrogenic, myogenic, and osteogeneic lineages.
  • the stromal vascular fraction including the mesenchymal stem cells contained therein, are useful in research and therapeutics.
  • a common source of adipose tissue, including the stromal vascular fraction, is
  • lipoaspirate the adipose tissue harvested from patients undergoing liposuction.
  • the lipoasperiate comprises adipocytes, fat, connective tissue, blood vessels, and stromal vascular fraction.
  • the adipose tissue is usually separated from non-adipose tissue using a tissue collection container that utilizes decantation, sedimentation, and/or centrifugation techniques to separate the materials.
  • the adipose tissue is then disaggregated using methods such as mechanical force (mincing or shear forces), enzymatic digestion with single or combinatorial proteolytic enzymes, such as collagenase, trypsin, lipase, liberase HI, pepsin, or a combination of mechanical and enzymatic methods.
  • mechanical force mincing or shear forces
  • enzymatic digestion with single or combinatorial proteolytic enzymes, such as collagenase, trypsin, lipase, liberase HI, pepsin, or a combination of mechanical and enzymatic methods.
  • a lipoaspirate may be obtained.
  • the fatty portion diluted with PBS and centrifuged to exclude all
  • Homogenization comprises both disruption and homogenization of the starting material as two distinct steps.
  • Disruption refers to the complete disruption of cell walls and plasma membranes of cells and organelles. Different samples require different methods to achieve complete disruption. Incomplete disruption results in significantly reduced yield.
  • Homogenization refers to the shearing of high-molecular-weight genomic DNA and other high-molecular-weight cellular components to create a homogeneous lysate. Incomplete homogenization results in significantly reduced yields.
  • Some disruption methods simultaneously homogenize the sample while others require an additional homogenization step.
  • Disruption and homogenization using rotor-stator homogenizers thoroughly disrupts and simultaneously homogenize, in the presence of lysis buffer, animal tissues in 5-90 seconds depending on the toughness of the sample.
  • Rotor-stator homogenizers can also be used to homogenize cell lysates. The rotor turns at a very high speed causing the sample to be disrupted and homogenized by a combination of turbulence and mechanical shearing. Foaming of the sample may be kept to a minimum by using properly sized vessels, by keeping the tip of the homogenizer submerged, and by holding the immersed tip to one side of the tube.
  • Rotor-stator homogenizers are available in different sizes and operate with differently sized probes. Probes with diameters of 5 mm and 7 mm are suitable for volumes up to 300 ⁇ and can be used for homogenization in microcentrifuge tubes. Probes with a diameter of 10 mm or above require larger tubes.
  • Disruption using a mortar and pestle is a classic method where one flash-freezes the sample immediately in liquid nitrogen and grinds to a fine powder in liquid nitrogen. Transfer the suspension (tissue powder and liquid nitrogen) into a liquid-nitrogen-cooled, appropriately sized tube and allow the liquid nitrogen to evaporate without allowing the sample to thaw. Add lysis buffer and continue as quickly as possible with the homogenization according to one of the methods below. Note that grinding the sample using a mortar and pestle will disrupt the sample, but it will not homogenize it. Homogenization must be performed separately before proceeding.
  • homogenization may be accomplished using spin-column homogenizers.
  • the lysate is loaded onto the spin column in collection tube and centrifuged.
  • the homogenized lysate is collected as flow-through.
  • beads-milling In disruption and homogenization using bead mills (“bead-milling"), cells and tissues can be disrupted by rapid agitation in the presence of beads. Some bead-milling method both disrupt and homogenize sample material in one step. Two different methods for disruption and homogenization using a bead-milling device. First, samples can be disrupted and homogenized at room temperature in lysis buffer or after freezing the sample-containing disruption vessel in liquid nitrogen. In the latter method, the lysis buffer is added after disruption of the sample. In special cases (e.g., the disruption of teeth or plant seeds), the sample can also be disrupted at room temperature without the addition of lysis buffer, although this increases the risk of nucleic acid degradation by nucleases.
  • Disruption and simultaneous homogenization occur by the shearing and crushing action of the beads as they collide with the sample. Disruption efficiency is influenced by: (a) size and composition of beads; (b) ratio of buffer to samples (if buffer is used); (c) composition of the lysis buffer; (d) amount of starting material; (e) configuration of agitator (speed and duration); (f) consistency of sample; and (g) type of disruption vessel.
  • the optimal beads to use are 0.1-0.6 mm (mean diameter) glass beads for bacteria, 0.5 mm glass beads for yeast and unicellular animal cells, and 3-7 mm stainless steel or tungsten carbide beads for plant and animal tissues. It is essential that glass beads are pretreated before use by washing in concentrated nitric acid.
  • QIAGEN News 2004 el l "Disruption and homogenization of starting materials for purification of RNA.” McGarvey, et al.
  • the invention provides a method of recovering a stromal vascular fraction from adipose tissue comprising providing adipose tissue obtained from an animal; mixing said adipose tissue with tumescent fluid; homogenizing said adipose tissue with beads for about 30 seconds to 6 minutes, wherein the adipose cells and blood vessels in the adipose tissue are lysed, thereby dissociating or releasing substantial numbers of intact stromal vascular fraction cells from the lysed blood vessels contained in the ultrasonicated adipose tissue while substantially maintaining the viability of the cells constituting the stromal vascular fraction.
  • the method may further comprise isolating the stromal vascular fraction (SVF), optionally removing the beads.
  • SVF stromal vascular fraction
  • the method may not include the addition of an endopeptidase, optionally collagenase.
  • the animal may be a mammal, optionally a human.
  • the adipose tissue may be obtained from the stromal or mesenchymal compartment of a human cadaver, tissue bank, organ donation, solid fat obtained from a human cadaver, or a liposuction derived aspirate.
  • the adipose tissue may be comminuted prior to homogenization with beads.
  • the animal may be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours post-mortem. In another embodiment, the animal may be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days post-mortem.
  • the adipose tissue may be comprised in phosphate buffered saline, normal saline, or another biologically acceptable liquid.
  • the beads may be stainless steel, zirconium oxide, tungsten carbide, ceramic, zirconium silicate, or glass beads, optionally zirconium oxide beads. In another embodiment, the beads may be about 0.01-2 mm beads, optionally 2 mm beads.
  • the adipose sample comprises about 7 mL of adipose tissue.
  • the adipose sample may be mixed with about 3 mL of tumescent fluid.
  • the adipose sample may be homogenized with beads for about 3 minutes.
  • the beads may be added at a bead-to-sample ratio of about 1: 1-1:4, optionally 1:4 (25% beads by volume).
  • the method may further comprise allowing the treated adipose tissue to settle or may be centrifuged, optionally for about 3 minutes at 500 RCF (relative centrifugal force), resulting in the fat rising to the top of the sample.
  • RCF relative centrifugal force
  • the stromal vascular fraction may comprise mesenchymal stem cells, hematopoietic cells, hematopoietic stem cells, platelets, Kupffer cells, osteoclasts,
  • megakaryocytes granulocytes, NK cells, endothelial precursor or progenitor cells, CD34+ cells or mesenchymal stem cells, CD29+ cells, CD 166+ cells, Thy-1+ stem cells, CD90+ stem cells, CD44+ cells, monocytes, leukocytes, lymphocytes, B cells, T cells, NK cells, macrophages, neutrophil leukocytes, neutrophils, and neutrophil granulocytes.
  • the sample may be assayed, optionally by flow cytometry, for the presence of adipose-derived stem cells including CD34 and/or Thy-1 or CD90 expressing stem cells.
  • the sample may be fractionated using fluorescence activated call sorting (FACS) based on cell surface antigens which may be specific to adipose-derived stem cells.
  • FACS fluorescence activated call sorting
  • the method may further comprise isolating the stromal vascular fraction and cryopreserving said stromal vascular fraction.
  • the method results in a yield of at least about lxlO 5 to lxlO 7 stromal vascular cells per mL of adipose tissue. In another embodiment, at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of said stromal vascular fraction cells isolated may be viable.
  • the method of recovering a stromal vascular fraction from adipose tissue may comprise providing about 7 mL of adipose tissue obtained from an animal, optionally lipoaspirate; mixing said adipose tissue with about 3 mL of tumescent fluid; homogenizing said adipose tissue with about 5 mL of 2 mm zirconium oxide beads for about 3 minutes at a bead-to- sample ratio of about 1:3 or about 1:4, wherein the adipose cells and blood vessels in the adipose tissue may be lysed, thereby dissociating or releasing substantial numbers of intact stromal vascular fraction cells from the lysed blood vessels contained in the ultrasonicated adipose tissue while substantially maintaining the viability of the cells constituting the stromal vascular fraction; and isolating the stromal vascular fraction (SVF) cells, optionally removing the beads.
  • stromal vascular fraction SVF
  • the invention provides an isolated stromal vascular fraction derived from adipose tissue by the method described herein, that does not comprise any exogenous collagenase.
  • the stromal vascular fraction comprises stem cells that express at least one protein selected from the group consisting of CD13, CD29, CD34, CD36, CD44, CD49d, CD54, CD58, CD71, CD73, Thy- 1, CD90, CD105, CD106, CD151, and SH3.
  • the stromal vascular fraction comprises stem cells that express at least one protein selected from the group consisting of CD31, CD45, CD117, and CD146.
  • the stromal vascular fraction comprises stem cells that do not express CD56.
  • the stromal vascular fraction comprises stem cells that do not express at least one protein selected from the group consisting of CD3, CD4, CD14, CD15, CD16, CD19, CD33, CD38, CD56, CD61, CD62e, CD62p, CD69, CD104, CD135, and CD144.
  • the stromal vascular fraction comprises stem cells that express CD49d but do not express CD56.
  • a method for cosmetic surgery may comprise administering a stromal vascular fraction obtained by the method described herein.
  • a use of the stromal vascular fraction obtained by a method described herein may be used in the manufacture of cosmetic products.
  • a method for tissue transplantation, optionally allogenic or autologous may comprise administering a stromal vascular fraction obtained by a method described herein.
  • the use of the stromal vascular fraction obtained by a method described herein may be used in tissue transplantation, optionally allogeneic or autologous transplantation.
  • the stromal vascular fraction may be used in a cosmetic surgery application, to promote wound healing, may be used in a tissue filler or in association with breast augmentation or reconstruction, tissue engineering, or burn treatment.
  • a method of treating a disease may comprise administering a stromal vascular fraction obtained by a method described herein.
  • the use of the stromal vascular fraction obtained by a method described herein may be used in the manufacture of a medicament for the treatment of a disease products.
  • the method or use may include treatment alone or in combination with tissue fillers, use for treating gum recession, treating loss of bone, including the jaw, treatment of orthopedic problems, treatment of arthritis, treatment of migraine, treatment of multiple sclerosis, treatment of autism, treatment of diabetes, treatment of wounds, treatment of ulcers, treatment of COPD, treatment of plantar fascitis, treatment of rotator cuff, and treatment of tennis elbow.
  • a method of recovering a stromal vascular fraction from adipose tissue may comprise providing about 7 mL of adipose tissue, optionally obtained from a nonliving animal; treating said adipose tissue homogenization for about 30-60 seconds with 2 mm zirconium oxide beads, wherein the adipose cells and blood vessels in the adipose tissue may be lysed, thereby dissociating or releasing substantial numbers of intact stromal vascular fraction cells from the lysed blood vessels contained in the adipose tissue while substantially maintaining the viability of the cells constituting the stromal vascular fraction.
  • the method may further comprise isolating the stromal vascular fraction (SVF).
  • the stromal vascular fraction cells may be cultured and/or expanded.
  • mesenchymal stem cells may be isolated from the stromal vascular fraction.
  • the mesenchymal stem cells may be cultured and/or expanded.
  • the method may not include the addition of an endopeptidase, optionally collagenase. In another embodiment, the method may include the addition of an endopeptidase, optionally collagenase.
  • the animal may be a mammal, optionally a human.
  • the adipose tissue may be obtained from the stromal or mesenchymal compartment of a human cadaver, solid fat obtained from a human cadaver, or a liposuction derived aspirate.
  • the animal may be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours post-mortem.
  • the animal may be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days post-mortem.
  • the adipose tissue may be obtained from a tissue bank, organ donation, or stored (e.g., frozen tissue).
  • the adipose tissue may be comprised in phosphate buffered saline, normal saline, or another biologically acceptable liquid.
  • the adipose sample may comprise about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 cc of adipose tissue.
  • the adipose sample may comprise about 7, 8, 9, or 10 cc of adipose tissue.
  • the adipose sample may comprise up to about 60 cc of adipose tissue or about 20-60 cc adipose tissue.
  • the method may further comprise allowing the treated adipose tissue to settle or may be centrifuged, optionally for about 3 minutes at 500 RCF (relative centrifugal force), resulting in the fat rising to the top of the sample.
  • RCF relative centrifugal force
  • the stromal vascular fraction may comprise mesenchymal stem cells, hematopoietic cells, hematopoietic stem cells, platelets, Kupffer cells, osteoclasts, megakaryocytes, granulocytes, NK cells, endothelial precursor or progenitor cells, CD34+ cells or mesenchymal stem cells, CD29+ cells, CD 166+ cells, Thy- 1+ stem cells, CD90+ stem cells, CD44+ cells, monocytes, leukocytes, lymphocytes, B cells, T cells, NK cells, macrophages, neutrophil leukocytes, neutrophils, and neutrophil granulocytes.
  • the sample may be assayed, optionally by flow cytometry, for the presence of adipose-derived stem cells including CD34 and/or Thy-1 or CD90 expressing stem cells.
  • the sample may be fractionated using fluorescence activated call sorting (FACS) based on cell surface antigens which may be specific to adipose-derived stem cells.
  • FACS fluorescence activated call sorting
  • the method may further comprise isolating the stromal vascular fraction and cryopreserving said stromal vascular fraction.
  • the method may result in a yield of at least about lxlO 4 to lxlO 7 stromal vascular cells per mL of adipose tissue. In another embodiment, the method may result in a yield of at least about l-5xl0 6 stromal vascular cells per mL of adipose tissue [0044] In a further embodiment, the method may comprise aliqouting the homogenized adipose tissue sample with equal amounts of 0.9% Sodium Chloride. In another embodiment, the homogenized adipose tissue/ 0.9% Sodium Chloride mixture may be centrifuged.
  • At least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of said stromal vascular fraction cells isolated may be viable.
  • the stromal vascular fraction may be located in blood vessels contained in or proximate to adipose tissue.
  • a method of recovering a stromal vascular fraction adipose tissue may comprise providing about 5-10 mL of adipose tissue, optionally obtained from a nonliving animal, in a 50 cc size tube; treating said adipose tissue with bead homogenization for 30-60 seconds with 0.1 mm beads, or from about 30 seconds to 6 minutes, wherein the adipose cells and blood vessels in the adipose tissue may be lysed, thereby dissociating or releasing substantial numbers of intact stromal vascular fraction cells from the lysed blood vessels contained in the homogenized adipose tissue while substantially maintaining the viability of the cells constituting the stromal vascular fraction.
  • the stromal vascular fraction may comprise stem cells that express at least one protein selected from the group consisting of CD 13, CD29, CD34, CD36, CD44, CD49d, CD54, CD58, CD71, CD73, Thy- 1, CD90, CD105, CD106, CD151, and SH3.
  • the stromal vascular fraction may comprise stem cells that express at least one protein selected from the group consisting of CD31, CD45, CD117, and CD146.
  • the stromal vascular fraction may comprise stem cells that do not express CD56.
  • the stromal vascular fraction may comprise stem cells that do not express at least one protein selected from the group consisting of CD3, CD4, CD14, CD15, CD16, CD19, CD33, CD38, CD56, CD61, CD62e, CD62p, CD69, CD104, CD135, and CD144.
  • the stromal vascular fraction may comprise stem cells that express CD49d but do not express CD56.
  • a method for cosmetic surgery may comprise administering a stromal vascular fraction obtained by the method described herein.
  • the use of the stromal vascular fraction obtained by the method described herein may be used in the manufacture of cosmetic products.
  • the stromal vascular fraction may be used in a cosmetic surgery application, to promote wound healing, may be used in a tissue filler or in association with breast augmentation or reconstruction, tissue engineering, or burn treatment.
  • a method of treating a disease comprising administering a stromal vascular fraction obtained by the method described herein.
  • the method and use may include treatment alone or in combination with tissue fillers, use for treating gum recession, treating loss of bone, including the jaw, treatment of orthopedic problems, treatment of arthritis, treatment of migraine, treatment of multiple sclerosis, treatment of autism, treatment of diabetes, treatment of wounds, treatment of ulcers, treatment of COPD, treatment of plantar fascitis, treatment of rotator cuff, and treatment of tennis elbow.
  • a method for allogenic transplant may comprise administering a stromal vascular fraction obtained by the methods described herein to a patient in need thereof.
  • a method for autologous transplant may comprise administering a stromal vascular fraction obtained by the methods described herein to a patient in need thereof, wherein the adipose tissue sample may be obtained from said patient.
  • a method for allograft may comprise administering a stromal vascular fraction obtained by the methods described herein to a patient in need thereof.
  • ASC or ADSC Adipose-Derived Stem Cell
  • adipose tissue e.g., CD34 expressing hematopoietic stem cells.
  • Adipose tissue-derived cell refers broadly to a cell that originates in adipose tissue, from the blood vessels contained therein.
  • the initial cell population isolated from adipose tissue is a heterogeneous cell population including, but not limited to, stromal or mesenchymal vascular fraction (SVF)..
  • SVF stromal or mesenchymal vascular fraction
  • Adipose tissue refers broadly to any fat tissue.
  • the adipose tissue may be brown or white adipose tissue.
  • the adipose may be mesenchymal or stromal.
  • the adipose tissue is subcutaneous white adipose tissue.
  • the adipose tissue may be from any organism having fat tissue.
  • the adipose tissue is mammalian, most preferably the adipose tissue is human.
  • a convenient source of human adipose tissue is that derived from liposuction surgery or other surgery.
  • Adipose tissue may be obtained from non-living donors, including animals, including mammals, post-mortem.
  • Adipose-derived stem cell refers broadly to stromal or mesenchymal cells that originate from blood vessels found in adipose tissue which can serve as stem cell-like precursors to a variety of different cell types including but not limited to adipocytes, osteocytes, chondrocytes, muscle and neuronal/glial cell lineages.
  • Adipose-derived stem cells make up a subset population derived from adipose tissue which can be separated from other components of the adipose tissue using standard culturing procedures or other methods disclosed herein.
  • adipose-derived adult stem cells can be isolated from a mixture of cells using the cell surface markers disclosed herein.
  • Adipose cell refers broadly to any type of adipose tissue, including an undifferentiated adipose-derived adult stem cell and a differentiated adipose-derived adult stem cell.
  • Allogeneic refers broadly to any material derived from a different mammal of the same species.
  • Applicator refers broadly to any device including, but not limited to, a hypodermic syringe, a pipette, for administering the compounds and compositions of the invention.
  • Autologous refers broadly to any material derived from the same individual to which it is later to be re-introduced.
  • Bead homogenization and “homogenization with bead mills,” as used herein, refers broadly to a process where cells and tissues are disrupted by rapid agitation in the presence of beads. This process may also be referred to as “bead milling.”
  • Central nervous system refers broadly to include brain and/or the spinal cord of a mammal. The term may also include the eye and optic nerve in some instances.
  • Cosmetically or aesthetically effective amount refers broadly to a compound or cells is that amount of compound or cells which is sufficient to provide a cosmetically or aesthetically beneficial effect to the subject to which the compound or cells are administered such as skin rejuvenation, enhancement in plumpness or volume or appearance of treated tissue such as the cheeks, lips, buttocks, or breast tissue. Also, as used herein, a
  • “cosmetically effective amount” is the amount of cells which is sufficient to provide a beneficial effect to the subject to which the cells are administered.
  • a differentiated cell refers broadly to a cell that has achieved a terminal state of maturation such that the cell has developed fully and demonstrates biological specialization and/or adaptation to a specific environment and/or function.
  • a differentiated cell is characterized by expression of genes that encode differentiation-associated proteins in that cell. For example expression of GALC in a leukocyte is a typical example of a terminally
  • “Differentiation medium,” as used herein, refers broadly to a cell growth medium comprising an additive or a lack of an additive such that a stem cell, adipose tissue derived stromal cell, embryonic stem cell, ES-like cell, MSCs, neurosphere, NSC or other such progenitor cell, that is not fully differentiated when incubated in the medium, develops into a cell with some or all of the characteristics of a differentiated cell.
  • “Differentiating,” as used herein, refers broadly to a cell that is in the process of being differentiated.
  • “Differentiated adipose-derived adult stem cell” refers broadly to an adipose-derived adult stem cell isolated from any adipose tissue that has differentiated as defined herein.
  • Effective amount refers broadly to a compound is that amount of compound or cells which is sufficient to provide a beneficial effect to the subject to which the compound is administered. Also, as used herein, an “effective amount” is the amount of cells which is sufficient to provide a beneficial effect to the subject to which the cells are
  • Endogenous refers broadly to any material from or produced inside an organism, cell or system.
  • exogenous refers broadly to any material introduced from or produced outside an organism, cell, or system. In particular exogenous may refer to a material that is not present in the treated adipose tissue.
  • Isolated refers broadly to material removed from its original environment in which it naturally occurs, and thus is altered by the hand of man from its natural environment. Isolated material may be, for example, exogenous nucleic acid included in a vector system, exogenous nucleic acid contained within a host cell, or any material which has been removed from its original environment and thus altered by the hand of man (e.g., "isolated cell”).
  • isolated refers broadly to a protein, cell, DNA, antibody, RNA, or biologically active portion thereof, that is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the biological substance is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
  • isolated cell refers broadly to a cell which has been separated from other components and/or cells which naturally accompany the isolated cell in a tissue or mammal.
  • Graft refers broadly to a cell, tissue or organ that is implanted into an individual, typically to replace, correct or otherwise overcome a defect.
  • a graft may further comprise a scaffold.
  • the tissue or organ may consist of cells that originate from the same individual; this graft is referred to herein by the following interchangeable terms: “autograft”, “autologous transplant”, “autologous implant” and “autologous graft”.
  • autograft autograft
  • autologous transplant autologous implant
  • autologous graft autologous graft
  • a graft comprising cells from a genetically different individual of the same species is referred to herein by the following interchangeable terms: “allograft”, “allogeneic transplant”, “allogeneic implant” and “allogeneic graft”.
  • a graft from an individual to his identical twin is referred to herein as an "isograft", a “syngeneic transplant", a “syngeneic implant” or a “syngeneic graft”.
  • a "xenograft” is referred to herein as an "isograft", a “syngeneic transplant", a “syngeneic implant” or a “syngeneic graft”.
  • xenogeneic transplant or “xenogeneic implant” refers to a graft from one individual to another of a different species.
  • Immunophenotype refers broadly to cell is used herein to refer to the phenotype of a cell in terms of the surface protein profile of a cell.
  • Late passaged adipose tissue-derived stromal cell refers broadly to a cell exhibiting a less immunogenic characteristic when compared to an earlier passaged cell.
  • the immunogenicity of an adipose tissue-derived stromal cell corresponds to the number of passages.
  • the cell has been passaged up to at least the second passage, more preferably, the cell has been passaged up to at least the third passage, and most preferably, the cell has been passaged up to at least the fourth passage.
  • mammals refers broadly to any and all warm-blooded vertebrate animals of the class Mammalia, including humans, characterized by a covering of hair on the skin and, in the female, milk-producing mammary glands for nourishing the young. Examples of mammals include but are not limited to alpacas, armadillos, capybaras, cats, camels,
  • Mammals include but are not limited to bovine, canine, equine, feline, murine, ovine, porcine, primate, and rodent species. Mammal also includes any and all those listed on the Mammal Species of the World maintained by the National Museum of Natural History, Smithsonian Institution in Washington DC.
  • Multipotential or “multipotentiality” refers broadly to the capability of a stem cell to differentiate into more than one type of cell.
  • Phenotypic characteristics refers broadly to mean at least one of the following characteristics: morphological appearance, the expression of a specific protein, a staining pattern or the ability to be stained with a substance.
  • Precursor cell "progenitor cell,” and “stem cell” are used interchangeably in the art and herein and refer broadly either to a pluripotent, or lineage-uncommitted, progenitor cell, which is potentially capable of an unlimited number of mitotic divisions to either renew itself or to produce progeny cells which will differentiate into the desired cell type.
  • pluripotent stem cells lineage-committed progenitor cells are generally considered to be incapable of giving rise to numerous cell types that phenotypically differ from each other.
  • progenitor cells give rise to one or possibly two lineage-committed cell types.
  • “Stromal vascular fraction,” as used herein, refers broadly to a cell fraction derived from blood vessels found in adipose tissue that comprises different cell types including by way of example mesenchymal stem cells, hematopoietic cells, hematopoietic stem cells, platelets, Kupffer cells, osteoclasts, megakaryocytes, granulocytes, NK cells, endothelial precursor or progenitor cells, CD34+ cells or mesenchymal stem cells, (typically found in umbilical cord), CD29+ cells, CD 166+ cells, Thy-1+ or CD90+ stem cells, CD44+ cells, immune cells such as monocytes, leukocytes, lymphocytes, B and T cells, NK cells, macrophages, neutrophil leukocytes, neutrophils, neutrophil granulocytes, and the like including immune and other cells that express one or more of the following markers: CD3, CD 14 (macrophage marker), CD 19, CD20 (B cell
  • Treat refers broadly to reduce the frequency of the disease or disorder reducing the frequency with which a symptom of the one or more symptoms disease or disorder is experienced by an animal.
  • Undifferentiated adipose-derived adult stem cell refers broadly to a cell isolated from adipose tissue and cultured to promote proliferation, but has no detectably expressed proteins or other phenotypic characteristics indicative of biological specialization and/or adaptation.
  • Xenogeneic refers broadly to any material derived from a mammal of a different species.
  • adipose tissues e.g., derived from surgical excision or aspirated via liposuction
  • adipose tissues may be treated by homogenization with beads (bead-milling) for a sufficient amount of time to lyse the fat cells and the blood vessels contained therein and thereby release stromal vascular fraction cells contained within the blood vessels in the adipose tissue including stromal and mesenchymal stem cells, endothelial precursor cells, and other cell types which constitute the stromal vascular fraction.
  • the invention provides a novel method of obtaining a stromal vascular fraction from adipose tissue that does not include the use of collagenase or other enzymes to digest the collagen bonds that hold together the tissue.
  • collagenase works well for this purpose, and used in the art to degrade collagen and separate the tissue into discrete cells
  • the use of this enzyme may be disadvantageous for cellular products that are to be used in humans, e.g., cells or cell fractions which are to be used in tissue reconstruction or regeneration, e.g., breast reconstruction procedures, cosmetic skin rejuvenation or usage in cosmetic tissue fillers that are used during plastic surgery.
  • the FDA may consider that the use of this enzyme (to derive desired cells) results in a "maximally manipulated" cellular product. This is
  • stromal vascular fraction containing mesenchymal stem cells, endothelial cells, and other cells found in adipose tissues
  • adipose tissue derived composition does not include collagenase and is not "maximally manipulated" according to the FDA.
  • adipose tissues e.g., derived from surgical excision or aspirated via liposuction
  • adipose tissues may be treated ex vivo by homogenization with beads for a sufficient amount of time to lyse the fat cells and the blood vessels contained therein thereby releasing the stromal vascular fraction cells contained within the outer layer of blood vessel walls contained in the adipose tissue including stromal and mesenchymal stem cells, endothelial precursor cells, and other cell types which constitute the "stromal vascular fraction”.
  • the present inventor has found that the treatment of adipose tissue by use of bead milling under appropriate conditions such as exemplified in the working examples, not only lyses the fat cells, but further lyses the blood vessels contained therein, without adversely affecting the viability of stromal and mesenchymal stem cells, thereby releasing high numbers of viable stromal and mesenchymal stem cells, endothelial precursor cells, and other cell types which constitute the "stromal vascular fraction" which stromal and mesenchymal stem cells may be recovered and used in desired cosmetic or therapeutic methods wherein these cells are of beneficial value.
  • post-mortem sources of adipose tissue may be used to obtain the stromal vascular fraction.
  • adipose tissue taken from an animal up to 28 days post-mortem the homogenization with beads method described herein resulted in a high yield of viable cells.
  • This surprising result has opened up an entirely new source of adipose tissue for processing to obtain a stromal vascular fraction for cosmetic, research, and therapeutic uses (e.g., post-mortem sources).
  • the adipose tissue treated by the claimed method described herein may be obtained from a variety of living and non-living sources including but not limited to animals (e.g., cows, chickens, sheep, goats, pigs) and humans (e.g., lipoaspirate, removed during surgery, or from cadavers).
  • animals e.g., cows, chickens, sheep, goats, pigs
  • humans e.g., lipoaspirate, removed during surgery, or from cadavers.
  • a good candidate for lipocavitation is someone looking for fat removal from a specific area such as the hips, thighs, buttocks, stomach or arms.
  • the treatment does not generally result in overall weight loss, but an improved contour in the localized treatment area.
  • the handpiece delivers low frequency ultrasound waves down into the subcutaneous or fatty layer of the skin, targeting the adipocytes or fat cells.
  • the minute vibrations produce tiny bubbles within the fat cells which disturb the outer membrane and allow tiny collections of fat to be expelled into the surrounding area, which then is removed via the body's natural energy and waste removal processes.
  • This selective destruction of fat cells does not interfere with adjacent structures such as blood vessels and nerves and is therefore a very safe treatment. Lipocavitation is a painless procedure, though for some people there may be a little discomfort associated with the noise during treatment which ceases when the handpiece is no longer in contact with the skin.
  • ultrasonic cavitation or lipocavitation is well known as a non invasive treatment for the reduction of localized fat deposits. This method is used for people who are dissatisfied with a certain area of fatty deposits but who do not want to undergo any invasive surgical treatment like liposuction. It is performed as a walk in, walk out treatment and there is no lengthy recovery period as with surgical fat removal.
  • the homogenization with beads methods described herein produce a stromal vascular fraction and specific cell types contained therein from adipose tissue surgically obtained from the stromal or mesenchymal compartment of the body of a donor or derived from a liposuction derived aspirate.
  • the adipose tissue may be obtained from any post-mortem animal, for example an animal that is at least about 24 hours post-mortem.
  • the adipose tissue may be obtained from a human cadaver.
  • the adipose tissue may be obtained from a tissue bank, organ donation, or stored (e.g., frozen tissue).
  • the adipose tissue sample homogenized with beads may be a lipoaspirate, adipose tissue sample removed during surgery, adipose tissue obtained from a non-living mammal, or adipose tissue obtained from a non-living animal.
  • the homogenization with beads methods described herein produce a stromal vascular fraction from adipose tissue, which method comprises homogenizing adipose tissue with beads under conditions whereby that the fat cells in the sample are lysed, and in addition under homogenization conditions whereby the blood vessels found in the fat are further lysed without adversely affecting the viability of stromal and mesenchymal stem cells contained therein.
  • homogenization with beads methods may be used in the absence of protease treatment to release the desired stromal vascular or mesenchymal vascular cells from the blood vessels found in the adipose tissue without adversely affecting the stromal and mesenchymal stem cells substantial lysis or degradation of the stromal and mesenchymal stem cells.
  • the methods will not include the addition of an enzyme that breaks down collagen such as a collagenase or other endopeptidase.
  • the homogenization with beads methods described herein mechanically treat adipose tissue ex vivo preferably in the absence of exogenous collagenase to lyse fat cells and the resultant sonically treated composition (from which the fat is removed) is then used to obtain a stromal vascular fraction which can be infused directly in patients in need thereof or it can be further processed to purify (and expand in culture if desired) desired cell types such as mesenchymal or stromal stem cells, endothelial cells, and other cells found in adipose tissue.
  • fractions and cells may be used in patients such as for tissue reconstruction, tissue regeneration, wound healing, allograft, allogenic transplant, tissue transplantation, breast augmentation or reconstruction, in tissue fillers for plumping areas that have lost fullness, such as via aging or because of disease such as the face, lips, and the buttocks.
  • a homogenization with beads device for about 1 second to 5 minutes ⁇ e.g., 10 seconds to 3 minutes) as to lyse most of the fat cells in the adipose tissue and blood cells under conditions that release the stromal vascular fraction containing stromal and mesenchymal stem cells, endothelial precursors and other cell types contained therein without adversely affecting the viability and number of these cells from different samples.
  • the adipose tissue sample may be treated with homogenization with beads for about 30-60 seconds using 0.1 mm beads.
  • the present invention results in the recovery of increased numbers of viable stromal and mesenchymal stem cells (about ten-fold more) from adipose samples relative to prior art methods using collagenase or other enzymes.
  • the lysed fat (at the top of the composition) may be removed and the remaining fraction further purified or assayed ⁇ e.g., by flow cytometry) for the presence of desired cell types including stem and endothelial precursor cells, immune cells, osteoclasts, hematopoietic stem cells, and other cell types disclosed herein.
  • the mesenchymal or stromal stem cells may be isolated from the sample such as by flow cytometry or may be fractionated into different cell types using fluorescence activated cell sorting (FACS) based on cell surface antigens which are specific to adipose-derived stem cells or other cell lineages contained in adipose tissue.
  • FACS fluorescence activated cell sorting
  • the homogenization with beads method described herein provides for a method of isolating a stromal vascular fraction derived from adipose tissue that does not comprise any exogenous collagenase or other exogenous enzymes.
  • the stromal vascular fraction may comprise stem and other cells that express at least one protein selected from the group consisting of CD13, CD14, CD29, CD31, CD34, CD36, CD44, CD45. CD49d, CD54, CD58, CD71, CD73, CD90, CD105, CD106, CD151 and SH3, or CD13, CD29, CD34, CD36, CD44, CD49d, CD54, CD58, CD71, CD73, CD90, CD105, CD106, CD151 and SH3 and/or CD31, CD45, CDl 17 and CD146. Further, the stromal vascular fraction may comprise stem and other cells that do not express CD56.
  • the stromal vascular fraction may comprise stem and other cells that express at least one protein selected from the group consisting of CD3, CD4, CD14, CD15, CD16, CD19, CD33, CD38, CD56, CD61, CD62e, CD62p, CD69, CD104, CD135 and CD144, and does not express CD3, CD4, CD14, CD15, CD16, CD19, CD33, CD38, CD56, CD61, CD62e, CD62p, CD69, CD104, CD135 and CD144. Also, the stromal vascular fraction may comprise stem and other cells that express CD49d but do not express CD56.
  • the homogenization with beads method described herein produce a stromal vascular fraction from adipose tissue by homogenizing adipose tissue with 0.1 mm beads for about 30-60 seconds under conditions whereby that the fat cells in the sample are lysed thereby releasing the stromal vascular or mesenchymal vascular cells from the adipose tissue and which preferably does not include the addition of an enzyme that breaks down collagen such as a collagenase or other endopeptidase.
  • the adipose tissue may be derived from any mammal, optionally a living or non-living donor.
  • the adipose tissue may be obtained via liposuction surgery, aspiration of fat after these procedures or isolated by other surgical methods.
  • the donor may be the same patient who is to be treated with the stromal or mesenchymal vascular fraction or cells derived therefrom or will be an allogeneic donor that is immune compatible with the treated individual.
  • the stromal vascular fraction cells isolated by the homogenization with beads methods described herein may be used in allogenic transplants.
  • Homogenization devices known in the art may be used.
  • One suitable selection is the Bullet Blender ® Blue which uses microcentrifuge tubes, the Bullet Blender ® Blue 5 which uses 5 mL tubes and the Bullet Blender ® Blue 50 which uses 50 mL tubes. Air CoolingTM is also included with the Bullet Blender ® Storm. These devices can process a wide range of sample sizes, 1 mL to 50 mL. The speed settings may be 1 or 4-6 on the Bullet Blender ® Blue 50 dial.
  • Other devices which may be used include Omni® Bead Ruptor Homogenizer from Omni International of Kennesaw GA, and TissueLyserll® from Qiagen®.
  • the exploded fat (at the top of the composition) may be removed and the remaining stromal or mesenchymal vascular fraction from the lysed blood vessels may be further purified and/or assayed (e.g., by flow cytometry) for the presence of desired cell types including stem and endothelial precursor cells.
  • FACS fluorescence activated call sorting
  • Cell contained therein and markers isolatable from adipose tissue include by way of example mesenchymal stem cells, hematopoietic cells, hematopoietic stem cells, platelets, Kupffer cells, osteoclasts, megakaryocytes, granulocytes, NK cells, endothelial precursor or progenitor cells, CD34+ cells or mesenchymal stem cells, (typically found in umbilical cord), CD29+ cells, CD 166+ cells, Thy-1+ or CD90+ stem cells, CD44+ cells, immune cells such as monocytes, leukocytes, lymphocytes, B and T cells, NK cells, macrophages, neutrophil leukocytes, neutrophils, neutrophil granulocytes, and the like including immune and other cells that express one or more of the following markers: CD3, CD14 (macrophage marker), CD19, CD20 (B cell marker),CD29 (integrin unit) CD31 (endo
  • the isolated stromal vascular fraction or isolated cells are preferably administered into a patient in need thereof.
  • the stem cells are used to promote wound healing, breast augmentation or reconstruction, tissue engineering, or other applications, for example allogeneic or autologous transplantation.
  • This fraction may comprise adipose- derived stem cells that express at least one protein selected from the group consisting of CD13, CD29, CD34, CD36, CD44, CD49d, CD54, CD58, CD71, CD73, CD90, CD105, CD106, CD151 and SH3, or CD13, CD29, CD34, CD36, CD44, CD49d, CD54, CD58, CD71, CD73, CD90, CD105, CD106, CD151 and SH3 and/or CD31, CD45, CD117 and CD 146 and will not express CD56.
  • adipose- derived stem cells that express at least one protein selected from the group consisting of CD13, CD29, CD34, CD36, CD44, CD49d, CD54, CD58, CD71, CD73, CD90, CD105, CD106, CD151 and SH3 and/or CD31, CD45, CD117 and CD 146 and will not express CD56.
  • the vascular fraction may comprise stem cells that express at least one protein selected from the group consisting of CD3, CD4, CD14, CD15, CD16, CD19, CD33, CD38, CD56, CD61, CD62e, CD62p, CD69, CD104, CD135 and CD144, and does not express CD3, CD4, CD14, CD15, CD16, CD19, CD33, CD38, CD56, CD61, CD62e, CD62p, CD69, CD104, CD135 and CD144 or expresses CD49d and does not express CD56.
  • stromal vascular fraction or isolated cells derived from adipose tissue may be used directly for treatment, alternatively the cells may be expanded in culture such that a single milliliter of tissue yields over 400,000 cells. Aust, et al. (2004) Cvtotherapy 6: 1-8.
  • Undifferentiated human adipocyte cells express a distinct immunophenotype based on flow cytometric analyses and, following induction, produce additional adipocyte specific proteins. Aust, et al (2004) Cvtotherapy 6: 1-8; Gronthos, et al. (2001) J. Cell Physiol. 189: 54-63;
  • Human adipose-derived adult stem cells (huASCs) display multipotentiality, with the capability of differentiating along the adipocyte, chondrocyte, myogenic, neuronal, and osteoblast lineages. Aust, et al. (2004) Cvtotherapy 6: 1- 8; Gronthos, et al. (2001) J. Cell Physiol.
  • the undifferentiated human adipocyte cells undergo adipogenesis as evidenced by the fact that between 30% to 80% of the cells, based on flow cytometric methods, accumulate lipid vacuoles, which can be stained for neutral lipid with Oil Red O dye. Halvorsen, et al. (2001) Metabolism 50: 407 ⁇ 113; Sen, et al. (2001) J. Cell. Biochem. 81 : 312-319. Further, the stromal vascular fraction isolated by the methods described herein may be cultured.
  • SVFCs can be obtained from any animal (alive or dead) so long as adipose stromal cells within the animal are viable.
  • Suitable tissue sources of SVFC's include, but are not limited to any fat-containing tissue, e.g., brown or white adipose tissue such as subcutaneous white adipose tissue.
  • human adipose tissue is obtained from a living donor using surgical excision or suction lumpectomy.
  • the adipose tissue may be obtained from a pre-selected region on the subject, i.e., inguinal, retroperitoneal and gonadal, or any combination thereof.
  • the separated ADSC-containing tissue optionally can be washed with any suitable physiologically-compatible solution, such as phosphate buffer saline (PBS) or normal saline. Using the exemplified methods, washing is not required.
  • PBS phosphate buffer saline
  • the middle layer includes the lattice and adipocyte aggregates.
  • the bottom layer or cell pellet which is produced after the treated composition is allowed to settle or is centrifuged and contains the stromal vascular fraction cells (SVFCs).
  • SVFCs stromal vascular fraction cells
  • the cellular fraction of the bottom layer may be infused into a subject or may be further concentrated into a pellet by any suitable method, e.g., centrifugation, and retained for further processing.
  • the isolated stromal vascualar faction may be centrifuged at 500 RCF (relative centrifugical force) for about 3 minutes.
  • the stromal vascular fraction (SVF) may be resuspended and can be further washed in physiologically compatible buffer, centrifuged, and resuspended one or more successive times to achieve greater purity.
  • the cells of the washed and resuspended pellet may also be plated.
  • Morphological, biochemical or molecular-based methods may be used to identify or isolate the cells in the stromal vascular fraction (SVF).
  • SVFCs are isolated based on cell size and granularity since SVFCs are small and agranular.
  • stem cells tend to have longer telomeres than differentiated cells, SVFCs can be isolated by assaying the length of the telomere or by assaying for telomerase activity.
  • SVFCs can be separated from the other cells of the pellet
  • Suitable markers include any of the markers disclosed in this application or any combination thereof.
  • the stem cells may be cultured without differentiation using standard cell culture media, referred to herein as control medium (e.g., DMEM, typically supplemented with 5-15% serum (e.g., fetal bovine serum, horse serum).
  • control medium e.g., DMEM
  • serum e.g., fetal bovine serum, horse serum
  • the stem cells can be passaged at least five times or even more than twenty times in this or similar medium without differentiating to obtain a substantially homogeneous population of SVFCs.
  • the SVFCs can be identified by phenotypic identification.
  • the cells are plated at any suitable density which may be anywhere from between about 100 cells/cm 2 to about 100,000 cells/cm 2 (such as about 500 cells/cm 2 to about 50,000 cells/cm 2 , or, more particularly, between about 1,000 cells/cm 2 to about 20,000 cells/cm 2 ).
  • SVFCs initially plated at lower densities at less than 500 cells/cm , or alternatively, less than about 300 cells/cm2 or alternatively, at less than 100 cells/cm 2
  • SVFCs initially plated at lower densities at less than 500 cells/cm , or alternatively, less than about 300 cells/cm2 or alternatively, at less than 100 cells/cm 2
  • the stem cells can be subcloned into a multi-well plate at a statistical ratio for facilitating placing a single cell into each well (e.g., from about 0.1 to about 1 cell/well or even about 0.25 to about 0.5 cells/well, such as 0.5 cells/well).
  • Cloning can be facilitated by the use of cloning rings. See MacFarland (2000) Methods in Cell Sci. 22:63-66.
  • clones can be obtained by permitting the cells to grow into a monolayer and then shielding one and irradiating the rest of the cells within the monolayer. The surviving cell then will grow into a clonal population.
  • plated cells can be diluted to a density of 10 cells/ml and plated on Nunclon 96-well plates (Nalge Nunc International). Only wells that contain a single cell at the outset of the culture period are assayed for colony formation. Clones are detectable by microscopy after 4 to 5 days.
  • An exemplary culture condition for cloning stem cells comprises about 213 F12 medium+20% serum (preferably fetal bovine serum) and about 113 standard medium that has been conditioned with stromal cells or 15% FBS, 1% antibiotic/antimycotic in F-12/DMEM
  • [1 : 1]) e.g., cells from the stromal vascular fraction of liposuction aspirate, the relative proportions can be determined volumetrically.
  • the homogenization with beads may be performed using stainless steel, zirconium oxide, tungsten carbide, ceramic, zirconium silicate, or glass beads.
  • the beads may be about 0.1 mm in diameter.
  • the beads may be about zirconium oxide beads 2 mm in diameter.
  • the beads may be about 0.01 to 2 mm in diameter.
  • the beads may be about 0.1, 0.2, 0.5, 1.0, 1.6, 2.0, 3.2, or 4.8 mm in diameter.
  • the beads may be a blend of 0.9-2.0 mm diameter beads.
  • the beads may have a diameter of about 0.05-2.0 mm.
  • the volume of beads may be about 10 mL to 25 mL. Also, the method may use 10-25 mL of 0.05-2.0 mm diameter beads for 30 seconds to about 6 minutes.
  • the beads may be added in a ratio of beads to sample of about 1: 1, 1: 1.2, 1: 1.5, 1: 1.75,
  • the beads may be added in a ration of beads to sample of about 1:4 (e.g.,
  • the adipose tissue sample size subjected to homogenization with beads may be about 1-
  • the adipose tissue sample size subjected to homogenization with beads may be about 4, 4.5, 5, 5.5, 6,
  • the adipose tissue may comminuted prior to homogenization with beads.
  • the adipose tissue may be mixed with tumescent fluid (a mixture of saline, Lidocaine®, and a vasoconstrictor, i.e., epinephrine).
  • tumescent fluid a mixture of saline, Lidocaine®, and a vasoconstrictor, i.e., epinephrine.
  • 7 mL of adipose tissue may be mixed with 3 mL of tumescent fluid for a total volume of 10 mL.
  • the homogenization with beads may be performed at a temperature at between about 0°C and 25°C.
  • the homogenization with beads may be performed at a temperature at about 4°C, for example in a cold room or using a cooling unit on the homogenizer ⁇ e.g., The Bullet Blender model with Air Cooling from Next Advance).
  • the homogenization with beads may be performed at a temperature at about 4°C, for example, using a Bullet Blender model with Air Cooling from Next Advance.
  • the homogenization with beads may be performed at a temperature about 0°C- 4°C.
  • the ability to perform the homogenization at a lower temperature ⁇ e.g., about 0°C ⁇ °C) contributes to the higher yield per mL of adipose tissue ⁇ e.g., less loss of viable cells to heat).
  • the adipose tissue may be homogenized with beads in a vessel of about 5 or 50 mL in size, preferably a conical polypropylene tube. Multiple tubes may be run in at a time ⁇ e.g., numerous tubes may be loaded into the homogenizer and homogenized in a single run).
  • the adipose tissue may be homogenized with beads at a speed of about 0.8m/s - 8m/s.
  • the speed setting for a Bullet Blender ® Blue 50 machine may be 4-6.
  • the homogenization with beads may be performed at a duration of about 1-60 seconds, 1-30 seconds, or 1-10 minutes.
  • the homogenization with beads may be for about 30-60 seconds.
  • the adipose tissue may be homogenized with beads for 10 seconds to 3 minutes.
  • the adipose tissue may be homogenized with beads for 30 seconds to 6 minutes.
  • the adipose tissue may be homogenized with beads for about 3 minutes.
  • the homogenization with beads may be performed by using a high-speed homogenizer with 0.1 mm beads.
  • the container may be a 50 cc tube size and the sample may be about 50 cc (mL).
  • the homogenization with beads method may result in a yield of about 200,000 up to about 22,000,000 stromal vascular cells per mL of adipose tissue.
  • the homogenization with beads method may result in a yield of about lxlO 5 , 2xl0 5 , 3xl0 5 , 4xl0 5 , 5xl0 5 , 6xl0 5 , 7xl0 5 , 8xl0 5 , 9xl0 5 , lOxlO 5 .
  • the homogenization with beads method may result in a yield of about lxlO 6 , 2xl0 6 , 3xl0 6 , 4xl0 6 , 5xl0 6 , 6xl0 6 , 7xl0 6 , 8xl0 6 , 9xl0 6 , lOxlO 6 , l lxlO 6 , 12xl0 6 , 13xl0 6 , 14xl0 6 , 15xl0 6 , 16xl0 6 , 17xl0 6 , 18xl0 6 , 19xl0 6 , 20xl0 6 , 21xl0 6 , or 22xl0 6 stromal vascular cells per mL of adipose tissue.
  • the homogenization with beads method may result in a yield of about lxlO 7 , 2xl0 7 , 3xl0 7 , 4xl0 7 , 5xl0 7 , 6xl0 7 , 7xl0 7 , 8xl0 7 , 9xl0 7 , or lOxlO 7 stromal vascular cells per mL of adipose tissue.
  • the homogenization with beads method may result in a yield of at least about lxlO 7 stromal vascular cells per mL of adipose tissue.
  • the homogenization with beads method may result in a cell yield with stromal vascular cells with at least about 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% viability.
  • the homogenization with beads method may result in a cell yield with stromal vascular cells with at least about 80%, 85%, 90%, or 95% viability.
  • the adipose tissue may be obtained from a non-living animal, including mammals.
  • the adipose tissue may be obtained from a mammal at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 hours post-mortem.
  • the adipose tissue may be obtained from a mammal at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 days post-mortem.
  • the adipose tissue may be obtained from a mammal including but not limited to alpacas, armadillos, capybaras, cats, camels, chimpanzees, chinchillas, cattle, dogs, goats, gorillas, hamsters, horses, humans, lemurs, llamas, mice, non-human primates, pigs, rats, sheep, shrews, squirrels, tapirs, and voles.
  • the adipose tissue may be harvested from a human.
  • the stromal vascular fractions and cells derived therefrom which are produced according to the invention have numerous applications including use in reconstructive and aesthetic plastic surgery, and therapies, especially indications wherein stem cells and differentiated cells derived therefrom have clinical or aesthetic efficacy. Because the subject methods avoid the use of collagenase or other substances which are undesired for infusion in humans the subject vascular fractions and cells contained therein may be directly infused into patients in need thereof.
  • the patient may be autologous, i.e., derived from the same donor or the cells may be infused into a compatible donor. Methods of HLA tissue matching cells for infusion into patients are well known in the art.
  • stromal vascular cells isolated from a homogenization with beads method described herein may be used for allogenic transplantation.
  • the homogenization with beads methods described herein mechanically treat adipose tissue ex vivo in the absence of collagenase to lyse fat cells and the blood cells contained therein and the resultant sonically treated composition (from which the fat is removed) is then used to obtain a stromal vascular fraction which can be infused directly in patients in need thereof or it can be further processed to purify (and expand in culture if desired) desired cell types such as mesenchymal stem cells, endothelial cells, and other cells found in adipose tissue.
  • tissue fillers for plumping areas that have lost fullness, such as via aging or because of disease such as the face, lips, the buttocks, and the like.
  • contemplated uses of these cells include use with or in lieu of tissue fillers, e.g., for treating gum recession, loss of bone, including e.g., the jaw, treatment of orthopedic problems, treatment of arthritis, treatment of migraine, treatment of multiple sclerosis, treatment of autism, treatment of diabetes, treatment of wounds, treatment of ulcers, treatment of COPD, treatment of plantar fascitis, treatment of rotator cuff, and treatment of tennis elbow.
  • the isolated mesenchymal or stromal stem cells or other cells are derived therefrom are infused or administered into a patient for a specific cosmetic or therapeutic procedure.
  • the isolated mesenchymal or stromal stem cells or other cells are derived therefrom are used to promote wound healing, breast augmentation or reconstruction, tissue engineering, or other applications.
  • Somatic tissue stem cells can be isolated from the subject stromal vascular fraction by fractionation using fluorescence activated call sorting (FACS) with unique cell surface antigens to isolate specific subtypes of stem cells (such as adipose derived stem cells) for injection into recipients following expansion in vitro, as described herein.
  • FACS fluorescence activated call sorting
  • Cells may be derived from the individual to be treated or a matched donor. Those having ordinary skill in the art can readily identify matched donors using standard techniques and criteria.
  • the stromal vascular fraction cells are vascular and reside in the walls of all blood vessels in the body and now that the method described herein can be used to harvest these cells from non-living adipose tissue.
  • the harvested cells can be stored in cryostorage based on their donor's blood type and be used allogenically with culturing.
  • the SVF may be stored and be readily available to treat patients for a multitude of injuries (e.g., orthopedic, post myocardiacal infarction).
  • the stromal vascular fraction cells stored may be matched to the patient by blood type and/or tissue type to ensure an allogeneic match.
  • the stromal vascular fraction cells may be stored at +4°C, -20°C, or -70°C.
  • the vascular cell fractions may be administered alone or in combination with tissue fillers (such as Juvederm) or scaffolds or matrices used to promote tissue regeneration or reconstruction, e.g., breast or other cancer reconstructive surgeries, foot surgery, breast augmentation, penile implants, facial fillers, joint or cartilage surgery, neck surgery, and the like.
  • tissue fillers such as Juvederm
  • scaffolds or matrices used to promote tissue regeneration or reconstruction, e.g., breast or other cancer reconstructive surgeries, foot surgery, breast augmentation, penile implants, facial fillers, joint or cartilage surgery, neck surgery, and the like.
  • the subject vascular cell fractions and stem cells derived therefrom may be used in cosmetic compositions used for topical application to the skin to effect rejuvenation and promote radiance, and reduce wrinkling.
  • the stromal vascular fraction produced according to the invention may be purified into desired cell types, e.g., a pure population of mesenchymal or stromal stem cells and these cells propagated in vitro using cell culture methods well known to those skilled in the art. As discussed herein those skilled in the art conventionally separate stem cells from other cells by FACS and other cell sorting methods based on the expression of characteristic markers.
  • the resultant purified stem cells may be injected into desired organs to effect tissue repair, e.g. into heart muscle to effect repair of the heart muscle, after a heart attack, into brain or spinal fluid to effect neural or nerve regeneration, such as Parkinsons or Alzheimers patients, into the bone or cartilage of individuals in need thereof such as individuals suffering from age, exertion, or disease related bone or cartilage loss.
  • tissue repair e.g. into heart muscle to effect repair of the heart muscle, after a heart attack
  • neural or nerve regeneration such as Parkinsons or Alzheimers patients
  • These purified stem cells may alternatively be cultured under conditions that give rise to desired cell lineages.
  • mesenchymal and stromal stem cells comprised in the subject fraction s may be differentiated into desired cell types including fibroblasts, neural cells, hematopoietic cells, myocytes, chondrocytes, and other cell types.
  • desired cell types including fibroblasts, neural cells, hematopoietic cells, myocytes, chondrocytes, and other cell types.
  • these cell types e.g., fibroblast populations may be seeded on a scaffold, which may be used in wound healing.
  • a system for separating and concentrating cells from adipose tissue in accordance with the invention may include one or more of a collection chamber, a processing chamber, a waste chamber, an output chamber and a sample chamber.
  • the various chambers are coupled together via one or more conduits such that fluids containing biological material may pass from one chamber to another in a closed, or functionally closed, sterile fluid/tissue pathway which minimizes exposure of tissue, cells, biologic and non-biologic materials with contaminants.
  • the waste chamber, the output chamber and the sample chamber are optional.
  • the system contains clinically irrelevant quantities of endotoxin.
  • the system also includes a plurality of filters.
  • the filters are effective to separate the stem cells and/or progenitor cells from, among other things, collagen, free lipids, adipocyte, that may be present in the solution after homogenization with beads of the adipose tissue sample.
  • the filter assembly may include a hollow fiber filtration device.
  • a filter assembly includes a percolative filtration device, which may or may not be used with a sedimentation process.
  • the filter assembly may comprise a centrifugation device, which may or may not be used with an elutriation device and process.
  • the system may comprise a combination of these filtering devices.
  • the filtration functions can be two-fold, with some filters removing things from the final concentration such as collagen, free lipid, free adipocytes, and with other filters being used to concentrate the final product.
  • One or more components of the system are automated and include an internal processing device and associated software programs which control many of the processing functions.
  • Components of the system may be disposable, such that portions of the system can be disposed of after a single use.
  • Such a system also comprises a re-usable component which includes the processing device (computer and associated software programs) and other components such as motors, pumps.
  • a method of treating a patient may comprise (a) providing a tissue removal system; (b) removing adipose tissue from a patient using the tissue removal system, the adipose tissue having a concentration of stem cells; (c) processing at least a part of the adipose tissue by use of homogenization with beads for a time sufficient to lyse all or most of the fat cells and release the mesenchymal and stromal vascular cells into a suitable fluid medium, e.g.
  • the active cell population is administered directly into the patient.
  • the active cell population e.g., the stem cells and/or endothelial precursor cells contained in the stromal vascular fraction
  • the active cell population are administered to the patient without being removed from the system or exposed to the external environment of the system before being administered to the patient.
  • Providing a closed system reduces the possibility of contamination of the material being administered to the patient.
  • processing the adipose tissue in a closed system provides advantages because the active cell population is more likely to be sterile.
  • the only time the stem cells and/or endothelial precursor cells are exposed to the external environment, or removed from the system is when the cells are being withdrawn into an application device and being administered to the patient.
  • the application device can also be part of the closed system.
  • the cells used may not processed for culturing or cryopreserved.
  • the active cells that have been concentrated, as described above, may be administered to a patient without further processing, or may be administered to a patient after being mixed with other tissues or cells.
  • the concentrated active cells e.g., stem cells or endothelial precursor cells
  • a composition comprising adipose tissue with an enhanced concentration of active cells may be administered to the patient.
  • the volumes of the various units of adipose tissue may be different. For example, one volume may be at least 25% greater than the volume of another unit of adipose tissue.
  • one volume may be at least 50%, such as at least 100%, and even 150% or more greater than the volume of another unit of adipose tissue.
  • the desired composition may be obtained by mixing a first unit of adipose tissue with the concentrated active cell population, which may be a cell pellet containing the active cells, with one or more other units of adipose tissue. These other units will not have an increased concentration of stem cells, or in other words, will have an active cell concentration less than that contained in the first unit of adipose tissue.
  • One of the units is cryopreserved material that contains, for example, an increased concentration of active cells.
  • At least a portion of the active cell population is stored for later implantation/infusion.
  • the population may be divided into more than one aliquot or unit such that part of the population of stem cells and/or endothelial precursor cells is retained for later application while part is applied immediately to the patient.
  • Moderate to long-term storage of all or part of the cells in a cell bank is also within the scope of this invention.
  • the cells may be mixed with one or more units of fresh or preserved adipose tissue to provide a composition containing the stem cells at a higher concentration than a unit of adipose tissue prior to processing.
  • the concentrated cells may be loaded into a delivery device, such as a syringe, for placement into the recipient by either subcutaneous, intravenous, intramuscular, or intraperitoneal techniques.
  • a delivery device such as a syringe
  • cells may be placed into the patient by any means known to persons of ordinary skill in the art, for example, they may be injected into blood vessels for systemic or local delivery, into tissue (e.g., cardiac muscle, or skeletal muscle), into the dermis (subcutaneous), into tissue space (e.g., pericardium or peritoneum), or into tissues (e.g., periurethral emplacement), or other location, such as placement by needle or catheter, or by direct surgical implantation in association with additives such as a preformed matrix.
  • tissue e.g., cardiac muscle, or skeletal muscle
  • tissue space e.g., pericardium or peritoneum
  • tissues e.g., periurethral emplacement
  • other location such as placement by needle
  • the active cell population may be applied alone or in combination with other cells, tissue, tissue fragments, demineralized bone, growth factors such as insulin or drugs such as members of the thiaglitazone family, biologically active or inert compounds, resorbable plastic scaffolds, or other additive intended to enhance the delivery, efficacy, tolerability, or function of the population.
  • the cell population may also be modified by insertion of DNA or by placement in cell culture in such a way as to change, enhance, or supplement the function of the cells for derivation of a cosmetic, structural, or therapeutic purpose.
  • gene transfer techniques for stem cells are known by persons of ordinary skill in the art, as disclosed in Mosca, et al.
  • Clin Orthop (379 Suppl): S71-90 may include viral transfection techniques, and more specifically, adeno-associated virus gene transfer techniques, as disclosed in Walther and Stein (2000) Drugs 60(2): 249-71, and Athanasopoulos, et al. (2000) Int J Mol Med 6(4): 363-75.
  • Non-viral based techniques may also be performed as disclosed in Muramatsu, et al. (1998) Int J Mol Med 1(1): 55-62.
  • the cells may be mixed with unprocessed fragments of adipose tissue and placed back into the recipient using a very large gauge needle or liposuction cannula. Transfer of autologous fat without supplementation with processed cells is a common procedure in plastic and reconstructive surgery.
  • Adipose tissue-derived cells of the invention that are, for example, substantially depleted of mature adipocytes may provide an environment that supports prolonged survival and function of the graft.
  • the cell population could be placed into the recipient and surrounded by a resorbable plastic sheath such as that manufactured by MacroPore Biosurgery, Inc. U.S. Patent Nos.
  • the sheath would prevent prolapse of muscle and other soft tissue into the area of a bone fracture thereby allowing the emplaced processed adipose tissue-derived cells to promote repair of the fracture.
  • the beneficial effect might be enhanced by supplementation with additional components such as pro-osteogenic protein growth factors or biological or artificial scaffolds.
  • the cells may be combined with a gene encoding a pro-osteogenic growth factor which would allow cells to act as their own source of growth factor during bone healing or fusion.
  • Addition of the gene could be by any technology known in the art including but not limited to adenoviral transduction, "gene guns,” liposome-mediated transduction, and retrovirus or Lentivirus-mediated transduction.
  • immunosuppressive agents may be administered to the patient receiving the cells and/or tissue to reduce, and preferably prevent, rejection of the transplant.
  • immunosuppressive agents suitable with the methods disclosed herein include agents that inhibit T-cell/B-cell costimulation pathways, such as agents that interfere with the coupling of T-cells and B-cells via the CTLA4 and B7 pathways, as disclosed in U.S. Patent Application Publication No.
  • 2002/018221 Other examples include cyclosporin, myophenylate mofetil, rapamicin, and anti- thymocyte globulin.
  • the cells are administered to a patient with one or more cellular differentiation agents, such as cytokines and growth factors.
  • cellular differentiation agents such as cytokines and growth factors.
  • cytokines and growth factors include IL-1, IL-2, IL-2, IL-4, IL-4, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12; and IL-12; and IL-12; and IL-12; and Cell differentiation agents, etc.
  • cytokines and growth factors are disclosed in Gimble, et al. (1995) J Cell Biochem 58(3): 393 ⁇ 102; Lennon, et al. (1995) Exp Cell Res 219(1): 21 1-22; Majumdar, et al. (1998) J Cell Physiol 176(1): 57-66; Caplan and Goldberg (1999) Clin Orthop (367 Suppl): SI 2-6; Ohgushi and Caplan (1999) J
  • stem cells and/or endothelial precursor cells By administering the stem cells and/or endothelial precursor cells to a patient, one can treat numerous diseases, including, and not limited to, bone-related disorders, diseases, or injuries, including slow/non-union fractures, osteoporosis (age-related or chemotherapy- induced), inherited diseases of bone (osteogenesis imperfecta); adipose related disorders or diseases; liver related diseases, disorders, or injuries, including liver failure, hepatitis B, and hepatitis C; myocardial infarctions, including heart attack or chronic heart failures; renal diseases or kidney damage; retinal diseases or damage or necrosis; wound healing (e.g., from surgery or diabetic ulcers); skeletal muscle disorders both traumatic and inherited; cartilage and joint repair both traumatic and autoimmune; lung injuries; diabetes; intestinal disorders; nervous system disorders, diseases, or injuries, such as central nervous systems disorders, diseases, or injuries, including spinal cord injuries, Parkinson's disease, Alzheimer's disease, and stroke.
  • the cells or composition containing may in addition further contain an additional pharmaceutical or agent, or alternatively a polynucleotide that encodes for a therapeutic agent or for an inhibiting nucleic acid.
  • additional pharmaceutical or agent or alternatively a polynucleotide that encodes for a therapeutic agent or for an inhibiting nucleic acid.
  • nuclear acids include, a ribozyme, an antisense oligonucleotide, a double stranded RNA, a double-stranded interfering RNA (iRNA), a triplex RNA, an RNA aptamer, and at least a portion of an antibody molecule that binds to the gene product and inhibits its activity.
  • the adipose tissue may be stored in liquid nitrogen (i.e., -70°C) or refrigerated (i.e., 4°C).
  • the stromal vascular fraction obtained by the homogenization with beads methods described herein may be stored in liquid nitrogen (i.e., -70°C) or refrigerated (i.e., 4°C). For example, one may harvest adipose tissue from cadaveric donors less than 24 hours post-mortem. SVF cells will be isolated from the cadaveric tissue and processed using the standard
  • the SVF cells will then be sorted/graded with immuno- affinity columns to isolate / remove allogenic antigens that would cause a transplant reaction.
  • the cells would be tested to match common blood types and Rh factors to recipients and then cryo-preserved and frozen to be stored for use by patients requiring cellular therapies in physician's offices and hospitals at future dates.
  • the subject adipose derived stromal vascular fraction or stem and endothelial precursor cells purified or derived therefrom may be induced to differentiate.
  • T U 2013/057007 adipose derived stromal vascular fraction or stem and endothelial precursor cells purified or derived therefrom
  • the cells obtained from the methods described herein may be used in conformable tissue implant for use in repairing or augmenting a tissue defect or injury site that may contain stem cells.
  • the tissue implant contains a tissue carrier matrix comprising a plurality of biocompatible, bioresorbable granules and at least one tissue fragment in association with the granules.
  • the cells obtained from the methods described herein may be used for repairing a damaged urinary tract tissue of a subject.
  • U.S. Patent No. 7,875,276 The cells obtained from the methods described herein may be used for repairing a damaged urinary tract tissue of a subject.
  • the cells obtained from the methods described herein may be used tissue scaffolds suitable for use in repair and/or regeneration of musculoskeletal tissue when implanted in a body.
  • tissue repair implant comprising: a tissue carrier matrix comprising a plurality of biocompatible,
  • bioresorbable granules and at least one tissue fragment in association with the tissue carrier matrix the at least one tissue fragment having an effective amount of viable cells that can migrate out of the tissue fragment and populate the tissue carrier matrix, wherein the tissue carrier matrix is in the form of an injectable suspension, and wherein an average maximum outer diameter of the granules is in a range of about 150 to about 600 ⁇ .
  • the cells obtained from the methods described herein may be used in a method of implanting stem or endothelial precursor cells into a body of a patient, said method comprising the steps of: providing a support structure, harvesting a polysaccharide-based modified biofilm from bacteria, attaching viable cells for implantation to the support structure with the
  • the cells obtained from the methods described herein may be used in an implantable biodegradable device containing a fibrous matrix, the fibrous matrix being constructed from fibers A and fibers B, wherein fibers A biodegrade faster than fibers B, fibers A and fibers B are present in relative amounts and are organized such that the fibrous matrix is provided with properties useful in repair and/or regeneration of mammalian tissue, and which may contain mesenchymal or stromal stem or endothelial precursor cells.
  • the cells obtained from the methods described herein may be induced to express at least one phenotypic characteristic of a neuronal, astroglial, hematopoietic progenitor, or hepatic cell and then used in therapy or tissue reconstruction.
  • the cells obtained from the methods described herein may be used in methods and compositions for directing adipose-derived stromal cells cultivated in vitro to differentiate into cells of the chondrocyte lineage.
  • the cells obtained from the methods described herein may be used therapeutic treatment of a number of human conditions and diseases including repair of cartilage in vivo.
  • the cells obtained from the methods described herein may be used in methods of making bioremodelable graft prostheses prepared from cleaned tissue material derived from animal sources.
  • the bioengineered graft prostheses of the invention are prepared using methods that preserve cell compatibility, strength, and bioremodelability of the processed tissue matrix.
  • the bioengineered graft prostheses are used for implantation, repair, or use in a mammalian host. These prostheses may contain mesenchymal or stromal stem or endothelial precursor cells.
  • the invention provides a lipo-derived stem cell substantially free of adipocytes and include treatment of use with or in lieu of tissue fillers, as a gum recession, loss of bone, including the jaw, treatment of orthopedic problems, treatment of arthritis, treatment of migraine, treatment of multiple sclerosis, treatment of autism, treatment of diabetes, treatment of wounds, treatment of ulcers, treatment of COPD, treatment of plantar fascitis, treatment of rotator cuff, and treatment of tennis elbow.
  • the stromal vascular fraction cells may be frozen for storage.
  • the stromal vascular fraction cells may be stored by any appropriate method known in the art ⁇ e.g., cryogenically frozen) and may be frozen at any temperature appropriate for storage of the cells.
  • the cells may be frozen at about -20°C, -80°C, -120°C, -130°C, -135°C, -140°C, -150°C, - 160°C, -170°C, -180°C, -190°C, -196°C, at any other temperature appropriate for storage of cells.
  • Cryogenically frozen cells may be stored in appropriate containers and prepared for storage to reduce risk of cell damage and maximize the likelihood that the cells will survive thawing.
  • the stromal vascular fraction cells may be cryopreserved immediately following differentiation, following in vitro maturation, or after some period of time in culture.
  • the stromal vascular fraction cells may also be maintained at room temperature, or refrigerated at, for example, about 4°C.
  • the stromal vascular fraction cells may be harvested, washed in buffer or media, counted, concentrated (via centrifugation), formulated in freezing media (e.g., 90% FBS/10% DMSO), or any combination of these steps.
  • the stromal vascular fraction cells may be seeded in several culture vessels and serially expanded. As the stromal vascular fraction cells are harvested and maintained in FBS at about 4°C while several flasks of stromal vascular fraction cells are combined into a single lot.
  • the stromal vascular fraction cells may be also washed with saline solution (e.g., DPBS) at least 1, 2, 3, 4, or 5 times.
  • saline solution e.g., DPBS
  • the information on the label may include the type of cell (e.g., stromal vascular fraction cells), the lot number and date, the number of cells (e.g., lxlO 6 cells/mL), the expiration date (e.g., recommended date by which the vial should be used), manufacture information (e.g., name and address), warnings, and the storage means (e.g., storage in liquid nitrogen).
  • type of cell e.g., stromal vascular fraction cells
  • the lot number and date e.g., the number of cells (e.g., lxlO 6 cells/mL)
  • the expiration date e.g., recommended date by which the vial should be used
  • manufacture information e.g., name and address
  • warnings e.g., storage in liquid nitrogen.
  • Cryopreserved stromal vascular fraction (SVF) cell preparations described herein may comprise at least about 50,000-100,000 stromal vascular fraction cells.
  • the cryopreserved stromal vascular fraction cell preparations may also comprise at least about 20,000-500,000 SVF cells.
  • the cryopreserved SVF cell preparations may comprise at least about 5,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 75,000, 80,000, or 100,000 SVF cells.
  • cryopreserved SVF cell preparations may comprise at least about 1,000, 2,000, 3,000, 4,000, 5,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 75,000, 80,000, 100,000, or 500,000 SVF cells.
  • the cryopreserved SVF cell preparations may comprise at least about 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, lxlO 4 , 2xl0 4 , 3xl0 4 , 4xl0 4 , 5xl0 4 , 6xl0 4 , 7xl0 4 , 8xl0 4 , 9xl0 4 , lxlO 5 , 2xl0 5 , 3xl0 5 , 4xl0 5 , 5xl0 5 , 6xl0 5 , 7xl0 5 , 8xl0 5 , 9xl0 5 , lxlO 6 , 2xl0 6 , 3xl0
  • cryopreserved SVF cell preparations may be mammalian SVF cells, including human SVF cells. T/US2013/057007
  • cryopreserved SVF cell preparations described herein may comprise at least about 50,000-100,000 SVF cells/mL.
  • the cryopreserved SVF cell preparations may also comprise at least about 20,000-500,000 SVF cells/mL.
  • the cryopreserved SVF cell preparations may comprise at least about 5,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 75,000, 80,000, and 100,000 SVF cells/mL.
  • the cryopreserved SVF cell preparations may comprise at least about 1,000, 2,000, 3,000, 4,000, 5,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 75,000, 80,000, 100,000, or 500,000 SVF cells/mL.
  • the cryopreserved SVF cell preparations may comprise at least about 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, lxlO 4 , 2xl0 4 , 3xl0 4 , 4xl0 4 , 5xl0 4 , 6xl0 4 , 7xl0 4 , 8xl0 4 , 9xl0 4 , lxlO 5 , 2xl0 5 , 3xl0 5 , 4xl0 5 , 5xl0 5 , 6xl0 5 , 7xl0 5 , 8xl0 5 , 9xl0 5 , lxlO 6 , 2xl0 6 , 3xl0 6 , 4xl0 6 , 5xl0 6 , 6xl0 6 , 7xl0 6 , 8xl0 6 , 9xl0 6 , lxlO 7 , 2xl0 7 , 3xl0
  • the SVF cells of the invention may be recovered from storage following
  • the SVF cells recovered from cryopreservation also maintain their viability and differentiation status. For example, at least about 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the SVF cells may retain viability and differentiation following cryopreservation.
  • the SVF cells of the invention may be cryopreserved and maintain their viability after being stored for at least about 1, 2, 3, 4, 5, 6, or 7 days.
  • the SVF cells of the invention may also be cryopreserved and maintain their viability after being stored for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 months.
  • the SVF cells of the invention may be cryopreserved and maintain their viability after being stored for at least about 1 , 2, 3, 4, 5, 6, or 7 years.
  • cryopreservation preparation comprising SVF cells may be substantially free of DMSO.
  • the invention also provides a method of cryopreserving stromal vascular fraction cells comprising (a) isolating stromal vascular fraction cells, (b) centrifuging said stromal vascular fraction cells, and (c) resuspending said stromal vascular fraction cells in 10% DMSO/90% FBS solution, optionally at least about 10 4 stromal vascular fraction cells per mL.
  • a method of cryopreserving stromal vascular fraction cells comprising (a) isolating stromal vascular fraction cells, (b) centrifuging said stromal vascular fraction cells, and (c) resuspending said stromal vascular fraction cells in 10% DMSO/90% FBS solution, optionally at least about 10 4 stromal vascular fraction cells per mL.
  • the present invention provides for a method of providing a stromal vascular fraction composition to a clinical site comprising (a) thawing vials of cryopreserved stromal vascular fraction cells, (b) resuspending the stromal vascular fraction cells in media, (c) centrifuging the stromal vascular fraction cells, (d) resuspending the stromal vascular fraction cells in media, (e) aliqouting the stromal vascular fraction cells into vials, and (f) transferring to the clinical site.
  • the resuspension and centrifugation steps may be repeated at least 1, 2, 3, 4, or 5 times.
  • the stromal vascular fraction product is transported to the clinical site within at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours of completion of step (e).
  • the vials may be labeled.
  • the present invention also provides a method for a providing stromal vascular fraction cell preparation for sale comprising (a) producing stromal vascular fraction cells and (b) preparing said stromal vascular fraction cell preparations for transfer to a customer.
  • the method may comprise cryopreserving the stromal vascular fraction cells.
  • the method comprises offering said stromal vascular fraction cell preparations for sale.
  • the method comprises advertising the stromal vascular fraction cell preparations.
  • Donor reimbursement costs may include the cost of fat harvesting in the case of cadaveric tissue donation.
  • Non-Patent Literature All publications (e.g., Non-Patent Literature), patents, patent application publications, and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All such publications (e.g., Non-Patent Literature), patents, patent application publications, and patent applications are herein incorporated by reference to the same extent as if each individual publication, patent, patent application publication, or patent application was specifically and individually indicated to be incorporated by reference.
  • adipose tissue was processed and derive a stromal vascular fraction containing stem cells from adipose tissue (e.g., collected from patients as taught in the examples). It is to be understood that the protocol is exemplary and that the specifics may be modified by a skilled artisan in order to further optimize. Using this protocol, the inventor has processed hundreds of samples with consistently good results. As disclosed herein, and substantiated by Millipore studies (See TABLE 2) the subject ultrasonication protocol results in about 10-fold more viable cells than comparable adipose samples treated with collagenase. Also, the inventive methods result in the same cell population and cell types as collagenase isolation procedures, suggesting that the inventive methods preserve the integrity of all the desired stromal vascular fraction cells, and especially the cell types identified herein.
  • metal infusion cannula attachment submerge to bottom of specimen tube and remove liquid stem cells solution including the pellet (from this sample take approximately 2cc of liquid to be used for testing with Flow Cytometer).
  • CD14 (monocyes that differentiate into a host of different cells) macrophages, neutrophils, granulocyte, dendretic cells
  • CD31 Endothelial cells endothelial cells, platelets, macrophages, granulocytes, TCells, natural killer cells, lymphocytes, megakaryocytes, osteoclasts, nutrophils,
  • CD34 Hematopoetic cells, endothelial progenitor cells, endothelial cells
  • CD29 Integrin protein unit involved in tissue repair and immune response
  • CD73 enzyme to catalyze lymphocyte differentiation
  • CD166 fibroblasts activated t-cells, activated monocytes, neurons, epithelial cells
  • CD3 activated T-lymphocytes
  • CD14 natural killer cells, neutrophil, macrophages, monocytes
  • CD 19 B cells
  • CD20 B cells
  • CD56 skeletal muscle, neuronse, glia
  • CD44 Receptor for hyaloronic acid involved in tissue repair found in connective tissue, epithelial tissue, and neural tissue
  • U.S. Patent Application Publication No. 2006/0051865 describes the use of ultrasonic methods to release adult stem cells from adipose tissue. See id., Example 2. When the inventor reproduced their methods and disclosed operating conditions they were ineffective, i.e., they yielded few stromal vascular fraction cells. As measured by flow cytometry, the
  • the 2006/0051865 protocol is a slight improvement at 700,000 cells/ml.
  • the inventor has been able to isolate 2,000,000 up to 22,000,000 cells/ml.
  • the claimed method provides a yield of four times up to thirty times as much as the 2006/0051865 protocol.
  • the present invention consistently produces results in very high numbers of viable stromal vascular fraction cells, which are well suited for use in cell therapy or cosmetic procedures.
  • Tumescent solution saline containing 0.0001% adrenalin
  • hypodermic fat layer hypodermic fat layer
  • cannulae having 2-3 mm of inner diameter (made of metal with aspirator) are used for the liposuction operation.
  • Liposuction operations are well known in the art, and for example, can be referred to in Safe Liposuction and Fat Transfer Rhoda Narins (Ed) Marcel Dekker, Inc (2005) and Textbook of Liposuction Hanke, et al. Informa Pic. (2007).
  • Aspirated fat is washed with saline. About five to ten liters of washed aspirate was generated, and the resultant adipose tissue derived cellular materials are used for derivation of stromal vascular fractions.
  • Fat tissue was obtained by surgery from human subjects who had given their informed consent. Separation was conducted with techniques well known in the art. Briefly, human fat tissue was aseptically separated from fat tissue suctioned from human subjects who had given 13 057007
  • adipose tissue derived cellular materials are used for derivation of stromal vascular fractions.
  • Fat tissue may be obtained from a human cadaver using methods known in the art.
  • the adipose tissue may be refrigerated ⁇ i.e., 4°C), frozen ⁇ i.e., -20°C), stored in liquid nitrogen ⁇ i.e., - 70°C).
  • Adipose tissue derived from liposuction aspirates or surgically as described in the previous example are placed in a suitable tube and a biologic solution if desired ⁇ e.g., phosphate buffered saline solution or normal saline solution) and the adipose tissue in the composition is placed contact with the ultrasonic probe of an ultrasonic cavitation device as described in the Materials and Methods section above.
  • a biologic solution e.g., phosphate buffered saline solution or normal saline solution
  • the Amplitude is set at about 50-100%, typically about 100%
  • Cycle 0.1- 1.0 and about 50 cc fat lipoaspirate is placed into a tube, 60 cc tube size, 28 mm diameter and 110 mm length and is treated by ultrasonic cavitation for about 10 minutes where at 5 minutes, the ultracaviation is stopped and the probe is adjust upward towards the middle of the sample and continued for the remaining 5 minutes using a 14 mm ultrasonic rod.
  • the device may be set at about 50-100% intensity and frequency of about 10-100% for about 5-60 minutes for about 50 cc of adipose tissue.
  • This treatment explodes the fat cells and thereby releases the stromal vascular fraction into the biologic solution, e.g., phosphate buffered or normal saline.
  • the biologic solution e.g., phosphate buffered or normal saline.
  • this treatment does not include the addition of collagenase or equivalent enzyme intended to break down collagen as cell dissociation is instead accomplished by ultrasonic sonication.
  • the resultant solution is allowed to settle over time or is treated by centrifugation.
  • the fat will float to the top.
  • This solution will contain the stromal vascular fraction at the bottom which includes adipose-derived stem cells, endothelial cell precursors and other cells and this fraction is uncontaminated by exogenous enzymes such as collagenases.
  • the fat containing supernatant may be discarded.
  • an aspirator may be used to carefully perform suction without damaging the cells. 2013/057007
  • the stromal vascular fraction containing stem cells recovered in Example 2 and using the Protocol above is characterized by known methods, e.g., flow cytometry or FACS, e.g., using antibodies that detect markers expressed on mesenchymal and stromal adipose derived stem cells. These methods will detect the presence of viable stem cells.
  • the protocols disclosed herein are exemplary and that the specifics may be modified by a skilled artisan in order to further optimize.
  • the applicant has processed over 200 samples with consistently good results.
  • the stem cells resulting therefrom have been used to treat patients.
  • the applicant has compared the stem cell containing cell samples derived according to the invention to those derived by conventional procedures (collagenase derived samples). More specifically, adipose -derived stem cell samples produced according to the invention were compared to those obtained in a study by Millipore. The comparison revealed that the inventive ultrasonic cavitation procedures result in the same cell population. Unexpectedly, the inventive procedure is much more efficient, i.e., it consistently results in about 10 times the number of cells for the same amount of fat.
  • the inventor compared three methods of isolating stromal vascular or mesenchymal vascular cells including lipoaspirate, the protocol of U.S. Patent Application Publication No. 2006/0051865, and the method described herein.
  • lipoaspirate yields about 500,000 cells/ml
  • the U.S. Patent Application Publication No. 2006/0051865 protocol is a slight improvement at 700,000 cells/ml.
  • the inventor isolated 2,000,000 up to 22,000,000 cells/ml. This was an unexpected result because sonication is considered in the art for lysing cells and the length of ultracaviation (i.e., 10 minutes) was unusual as compared to what was tried in the art.
  • Chicken fat was purchased from the butcher. The chicken was killed the day before and kept on ice chips. The fat was exposed to ultrasonic cavitation protocol described in Example 1 and the Stromal Vascular Fraction counts were as follows: Viability: 86.5%, Cell Count: 1.28 x 10 7 cells per mL, and Debris: 5.57%. 7
  • Beef fat was purchased from the butcher. It is estimated that it was aged for
  • the ultrasonic cavitation method described herein may be used on post-mortem sources of adipose tissue to isolate a stromal vascular fraction with high cell yields and high cell viability.
  • Stromal vascular fraction cells may be isolated from adipose tissue by means of homogenization with beads.
  • a stored lipoaspirate sample from a human donor was defrosted and aliquoted into tube samples containing 7 mL of adipose tissue and 3 mL of tumescent fluid (a mixture of saline, Lidocaine®, and a vasoconstrictor, i.e., epinephrine).

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Abstract

L'invention concerne des procédés de traitement à l'aide de tissus adipeux, en utilisant l'homogénéisation avec des perles, afin de dissocier les cellules graisseuses et les vaisseaux sanguins contenus dans les tissus adipeux, pour obtenir ainsi des fractions vasculaires stromales à utiliser chez des patients humains. Ces procédés n'incluent de préférence pas l'utilisation d'enzymes de dissociation exogènes quelconques, tels que la collagénase, et entraînent des nombres accrus de cellules constituant les fractions vasculaires stromales (environ 10 fois plus) que les procédés qui utilisent la collagénase pour isoler ces cellules. De plus, les tissus adipeux peuvent être isolés et obtenus à partir d'animaux et de sources non vivantes (par ex. post-mortem).
PCT/US2013/057007 2012-08-28 2013-08-28 Isolation de fraction vasculaire stromale des tissus adipeux, obtenue à l'aide de l'homogénéisation avec des perles WO2014036094A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015005871A1 (fr) * 2013-07-10 2015-01-15 Agency For Science, Technology And Research Méthode d'isolement de fraction stroma-vasculaire
WO2016199149A1 (fr) * 2015-06-10 2016-12-15 The Medical Research, Infrastructure, And Health Services Fund Of The Tel Aviv Medical Center Appareil mécanique et procédé d'isolement de la fraction stroma-vasculaire
DE102015119877A1 (de) 2015-11-17 2017-05-18 Technische Universität Berlin Verfahren zur Herstellung eines Hautäquivalents sowie dessen Verwendung für in vitro und in vivo Transplantate
CN108410711A (zh) * 2017-11-09 2018-08-17 上海美致臻生物医学科技有限公司 一种全自动脂肪核心集群细胞、脂肪基质血管组分采集仪
EP3193893A4 (fr) * 2014-09-16 2018-09-05 Virginia Tech Intellectual Properties, Inc. Compositions de cellules souches, systèmes et utilisations associées

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Publication number Priority date Publication date Assignee Title
WO2015005871A1 (fr) * 2013-07-10 2015-01-15 Agency For Science, Technology And Research Méthode d'isolement de fraction stroma-vasculaire
EP3193893A4 (fr) * 2014-09-16 2018-09-05 Virginia Tech Intellectual Properties, Inc. Compositions de cellules souches, systèmes et utilisations associées
WO2016199149A1 (fr) * 2015-06-10 2016-12-15 The Medical Research, Infrastructure, And Health Services Fund Of The Tel Aviv Medical Center Appareil mécanique et procédé d'isolement de la fraction stroma-vasculaire
CN107921182A (zh) * 2015-06-10 2018-04-17 特拉维夫医学研究、基础设施和卫生服务医疗中心基金会 用于分离基质血管部分的机械仪器和方法
CN107921182B (zh) * 2015-06-10 2022-09-02 特拉维夫医学研究、基础设施和卫生服务医疗中心基金会 用于分离基质血管部分的机械仪器和方法
US11891627B2 (en) 2015-06-10 2024-02-06 The Medical Research, Infrastructure, And Health Services Fund Of The Tel Aviv Medical Center Mechanical apparatus and method for isolating stromal vascular fraction
DE102015119877A1 (de) 2015-11-17 2017-05-18 Technische Universität Berlin Verfahren zur Herstellung eines Hautäquivalents sowie dessen Verwendung für in vitro und in vivo Transplantate
WO2017084997A1 (fr) 2015-11-17 2017-05-26 Technische Universität Berlin Procédé de fabrication d'un équivalent de peau et son utilisation pour des essais in vitro et des greffes in vivo
CN108410711A (zh) * 2017-11-09 2018-08-17 上海美致臻生物医学科技有限公司 一种全自动脂肪核心集群细胞、脂肪基质血管组分采集仪

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