WO2020130800A1 - Milieu conditionné de cellules souches - Google Patents

Milieu conditionné de cellules souches Download PDF

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WO2020130800A1
WO2020130800A1 PCT/MY2019/050124 MY2019050124W WO2020130800A1 WO 2020130800 A1 WO2020130800 A1 WO 2020130800A1 MY 2019050124 W MY2019050124 W MY 2019050124W WO 2020130800 A1 WO2020130800 A1 WO 2020130800A1
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composition
cells
extract
phase
stem cells
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PCT/MY2019/050124
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Kunasekaran WIJENTHIRAN
Siew Ee LEE
Subramaniam AVINAASH
Li Jin TAN
Bin Khairuzzaman SHAH RIZAL
Somasundram CHANDRAN
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Cytonex Sdn. Bhd.
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Publication of WO2020130800A1 publication Critical patent/WO2020130800A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
    • A61K8/981—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin of mammals or bird
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61K35/33—Fibroblasts
    • 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
    • A61K35/48—Reproductive organs
    • A61K35/51—Umbilical cord; Umbilical cord blood; Umbilical stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/44—Oils, fats or waxes according to two or more groups of A61K47/02-A61K47/42; Natural or modified natural oils, fats or waxes, e.g. castor oil, polyethoxylated castor oil, montan wax, lignite, shellac, rosin, beeswax or lanolin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
    • A61K8/04—Dispersions; Emulsions
    • A61K8/042—Gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
    • A61K8/11—Encapsulated compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/92—Oils, fats or waxes; Derivatives thereof, e.g. hydrogenation products thereof
    • A61K8/922—Oils, fats or waxes; Derivatives thereof, e.g. hydrogenation products thereof of vegetable origin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/16—Emollients or protectives, e.g. against radiation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/18—Antioxidants, e.g. antiradicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations

Definitions

  • the invention relates to stem cell conditioned media, for use in clinical and cosmetic applications.
  • Stem cell conditioned media is media in which stem cells have been grown, such media containing various extracellular components from the stem cells e.g. growth factors which can be used to stimulate skin regeneration, hair growth, and the like.
  • An aim of the invention therefore is to provide improved stem cell conditioned media which overcome the above issues.
  • composition comprising:
  • the extracellular factors are encapsulated by the carrier oil.
  • the carrier oil protects the extracellular factors from oxidation and improves skin absorption.
  • the growth factors therein may be stable for up to two years at room temperature.
  • the extracellular factors from the stem cell conditioned medium comprises any or any combination of growth factors, proteins, soluble proteins, peptides, cytokines, chemokines, hormones, enzymes, interleukins, interferons, prostaglandins, microvesicles, exosomes, regulatory factors, anti-inflammatory factors signaling molecules and/or microRNA (miRNA).
  • miRNA microRNA
  • the growth factors include any or any combination of Epidermal Growth Factor (EGF), Prostaglandin E2 (PGE2), Transforming growth factor beta (TGF-b), Vascular Endothelial Growth Factor (VEGF), hepatocyte Growth Factor (HGF), Stromal Cell-derived Factor 1 (SDF-1), Interleukin- 10 (IL-10), Angiogenin-1 (Ang-1), Fibroblast Growth Factor (FGF), Platelet Derived Growth Factor (PDGF), Interleukin 1 receptor antagonist (IL-IRA), Interleukin 4 (IL-4), Interleukin 5 (IL-5), Interleukin 10 (IL-10), Interleukin 13 (IL-13), Interferon alpha (IFN-a), Eotaxin, Monocyte Chemoattractant Protein 1 (MCP-1), Chemokine (C-X-C motif) ligand 9 (CXCL9), Chemokine (C-C motif) ligand 3 (CCL3), Chemokine (C-C motif) ligand
  • the medium also contains a significant amount of 9-octadecenoic acid, a form of Oleic acid that is released by the stem cells.
  • 9-Octadecenoic acid is an unsaturated fatty acid that is the most widely distributed and abundant fatty acid in nature. It is used commercially in the preparation of oleates and lotions, and as a pharmaceutical solvent. This invention shows that human stem cells can produce this fatty acid, which aids in the administration of the factors to be used in creams, lotions and anti -ageing serums.
  • the stem cell conditioned medium was used to grow mesenchymal stem cells.
  • mesenchymal stem cells are derived from any or any combination of bone marrow, adipose tissue, umbilical cord, dental pulp, peripheral blood, amniotic fluid and/or endometrium.
  • the stem cell conditioned medium was lyophilised after the stem cells were removed therefrom.
  • the cells are removed after two or three passages, preferably two passages.
  • the composition further comprises extracellular factors from mature fibroblast cells.
  • the fibroblast cells are derived from foreskin or fetal fibroblast.
  • the extracellular factors from the mature fibroblast cells comprises exosomes and/or microvesicles.
  • the exosomes contain miRNA to increase proliferation of skin cells.
  • the mature fibroblast cells contain the message (in the form of miRNA) to direct production of skin cells, whereas the growth factors from the stem cells stimulate the production of skin according to the message, such that in combination wound healing by skin regeneration is enhanced.
  • the carrier oil is hypoallergenic.
  • the carrier oil is high in unsaturated fatty acids to increase skin absorption, such as coconut oil.
  • the composition comprises carboxymethylcellulose to increase skin absorption.
  • the composition is formulated as a cream for cosmetic applications, reducing wrinkles, increasing dermal hydration, increasing collagen synthesis, treating stretch marks, preventing UV-induced skin damaged and the like.
  • the composition is injectable for use in treating chronic diabetic wounds, ulcers, and the like.
  • a method for healing wounds by applying a composition as herein described.
  • first and second culture media are mixed together prior to being lyophilised.
  • the formulation containing 0.9% NaCl solution that is used in therapeutic injectables for the treatment of chronic wounds.
  • an oil-based formulation contains natural carrier oil, namely Babassu oil, Jojoba oil, Argan oil and non-natural carrier oils, namely mineral oils, caprylic triglyceride, isopropyl myristate and white oil. Phenoxyethanol is used as a preservative. Aloe vera gel is used as an excipient.
  • a water-based formulation contains natural carrier oil, namely Squalane and Camellia Japonica Seed Oil.
  • EDTA added in the water phase is used as a chelating agent to chelate metals present in the formulation thus stabilize growth factors present in the formulation.
  • Sodium Hyaluronate, Galactomyces Ferment Filtrate, Glycerine, Dipropylene Glycol Sodium PCA, and Algae extracts are added to the formulation as hydrating agents to increase the moisture content of the skin, when combined with growth factors, can result in a positive synergistic effect on skin moisture level by reducing the transepidermal water loss (TEWL).
  • a tablet formulation for sublingual purposes contains Sodium starch glycolate, microcrystalline cellulose, Magnesium stearate, Manitol and Saccharine.
  • a microcapsulization formulation contains EDTA and the essential oils from the oil-based formulation.
  • Figure 1 illustrates the morphology of cells as herein described: (a) Wharton’s Jelly stem cells (WJSC); (b) stem cells from human exfoliated deciduous teeth (SHED); (c) foreskin derived fibroblasts (FSF); (d) fetal fibrobast.
  • Figure 2 illustrates the summary of proteome mapping results.
  • Figure 3 is a flow chart for the exosome purification procedure based on differential ultracentrifugation.
  • Figure 4 illustrates the volume statistic table (by intensity) for particle size distribution assay.
  • Figure 5 illustrates the microscopic views and appearance of growth factors from SHED and WJSC mixed with different carriers: (a) ddH 2 0 + Mineral Oil; (b) Carboxymethyl Cellulose (CMC) + Mineral Oil; (c) Mineral Oil + CMC; (d) dd3 ⁇ 40 + Coconut Oil; (e) CMC + Coconut Oil; (f) Coconut Oil + CMC.
  • CMC Carboxymethyl Cellulose
  • Figure 6 is a graph illustrating growth kinetics.
  • Figure 7 illustrates a cell surface antigen study of WJSCs.
  • Figure 8 illustrates an immunomodulation study of WJSCs.
  • Figure 9 illustrates testimonial images of diabetic foot ulcers before and after treatment with the composition according to the invention.
  • Figure 10 illustrates testimonial images of skin rejuvenation before and after treatment with the composition according to the invention.
  • Figure 11 illustrates testimonial images of (a) sacral pressure ulcer; (b) diabetic wound ulcer; and (c) hip pressure ulcer before and after treatment with NaTH Plus regenerating gel.
  • MSC Human Mesenchymal stem cells
  • adipose tissue derived from bone marrow, adipose tissue, umbilical cord, dental pulp, peripheral blood, amniotic fluid, endometrium and any other cell type either on its own or in combination, that secrete growth factors, proteins, soluble proteins, peptides, cytokines, chemokines, hormones, enzymes, interleukins, interferons, prostaglandins, microvesicles, exosomes, regulatory and anti-inflammatory factors and signaling molecules and microRNA (miRNA).
  • miRNA microRNA
  • Human mesenchymal stem cells used are obtained from a single donor or from multiple donors. The cells can either be used immediately upon harvesting at Passage 2 or Passage 3 or after being cryopreserved for a period of time.
  • Growth factors proteins, soluble proteins, peptides, cytokines, chemokines, hormones, enzymes, interleukins, interferons, prostaglandins, microvesicles, exosomes, regulatory and anti-inflammatory factors and signaling molecules and microRNA (miRNA) are factors that have a desirable effect on the cells of the human body as a result of paracrine interactions.
  • the purpose of the invention is the production process, content and application of a stem cell conditioned medium for wound healing, in order to treat chronic diabetic wounds, for aesthetic applications and for cosmetic usage in skin rejuvenation and regeneration.
  • the production procedure is as follows:
  • the umbilical cord was taken out from the transport buffer and put onto a petri dish by using forceps.
  • the umbilical cord was cut into 3 segments on the petri dish.
  • Step 4 and 5 were repeated twice.
  • the umbilical cord was transferred into the tube filled with Dulbecco’s Phosphate- buffered saline without calcium and magnesium (DPBS (-,-)) each.
  • the umbilical cord was taken out and put onto the petri dish.
  • the arteries (Wharton’s Jelly is in between the arteries; since vein contains HUVEC- human umbilical vein endothelial cells, don’t remove the vein) were removed using forceps and surgical scissors.
  • the umbilical cord was transferred into the tube filled with 100% IPA for 10 to 20 seconds, preferably 15 seconds. Then, the umbilical cord was transferred into the tube filled with ddH 2 0 for another 10 to 20 seconds, preferably 15 seconds. This is to remove any organic substances.
  • the umbilical cord was then transferred into tube filled with dPBS
  • the umbilical cord was transferred into the tube filled with DMEM-KO and collagenase type 1 to soften the tissue using enzymatic/chemical digestion.
  • the umbilical cord was minced using scissors (mechanical digestion).
  • the tissues were incubated at 28 °C, 29 °C, 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, 36 °C, 37 °C or 38 °C, with 4%, 5% or 6% C0 2 , preferably at 37 °C, 5% C0 2 , for 18 hours.
  • CCM Cell culture medium
  • the tube was centrifuged at 1,600 x g, 1,700 x g, 1,800 x g, 1,900 x g or 2,000 x g for 8- 12 minutes, preferably 1,800 x g for 10 minutes.
  • the tissues were seeded into T75 pre-added with CCM (7mL each), with approximately 10 fragments in each flask.
  • SAMPLE COLLECTION 1 The extracted deciduous tooth was kept in a specimen container filled with normal saline to prevent dryness of the tooth before the cutting procedure that commences as soon as possible.
  • the extracted tooth was sterilized externally by using povidone iodine (PVP-I, Sigma Aldrich, St Louis, USA) and washed with distilled water to minimize the contamination from the tooth surface.
  • PVP-I povidone iodine
  • the tooth was held tightly by using a hard tissue cutter device.
  • the tooth was sectioned horizontally at the cementoenamel junction by using a sterilized diamond disc on a straight handpiece to expose the pulp and the dental pulp tissue was extirpated under an aseptic condition.
  • the dental pulp tissue was transferred into 1.5 mL tube containing DMEM KnockOut Basal Media (Gibco, Grand Island, NY).
  • the dental pulp tissue was washed three times (serial washing) in a washing buffer solution (2mL each well) that contains Dulbecco’s Phosphate-Buffered Saline without calcium and magnesium (DPBS -Ca 2+ , -Mg 2+ , Invitrogen, USA), 0.5% v/v of gentamicin and 0.5% v/v of antibiotic-antimycotic.
  • DPBS -Ca 2+ , -Mg 2+ , Invitrogen, USA 0.5% v/v of gentamicin and 0.5% v/v of antibiotic-antimycotic.
  • the tissue was transferred into 1.5 mL tube (pre-added with 500 m ⁇ of 3mg/mL Collagenase Type I).
  • the tissue was minced into small fragments using sterile scissors.
  • the tissue was incubated at 28 °C, 29 °C, 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, 36 °C, 37 °C or 38 °C, 4%, 5% or 6% CO2, for 20-40 minutes, preferably at 37 °C, 5% CO2 for 30 minutes.
  • CCM cell culture medium
  • the tube with tissue was centrifuged at 1,000 x g, 1,100 x g, 1,200 x g, 1,300 x g or 1,400 x g for 4-8 minutes, preferably at 1200 x g for 6 minutes.
  • Tissue fragments were seeded in a T-25 culture flask (pre-added and pre-warmed with 2mL of CCM) and incubated at 28 °C, 29 °C, 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, 36 °C, 37 °C or 38 °C, 4%, 5% or 6% C0 2 , preferably at 37 °C, 5% C0 2.
  • Human foreskin was obtained form 9-15 years old males during circumcision.
  • the foreskin was placed into a sterile 50 mL polypropylene conical centrifuge tubes pre-added with 25 mL of transport buffer stored at 4 °C, 5 °C, 6 °C, 7 °C, 8 °C, 9 °C or 10 °C. This entire process should be done in a room with temperature ranging from 20 °C to 25 °C and the foreskin must be placed into the transport buffer solution within 24 hours to maintain tissue viability.
  • the foreskin tissue was transferred into a 50 mL polypropylene conical centrifuge tube pre-added with 30 mL sterilized distilled water (SDW).
  • SDW sterilized distilled water
  • the sample was washed by inverting the tubes repeatedly to remove traces of blood and to prevent any contamination. This step was repeated at least three times to ensure that the foreskin is thoroughly washed.
  • the foreskin was immersed in 70% ethanol for 25 to 30 seconds to sterilize the surface and then immediately immersed into SDW for another 25 to 30 seconds to remove ethanol residues to create an immediate hypotonic environment on the skin’s surface to eliminate microbes.
  • the tissue was transferred into a 50 mL polypropylene conical centrifuge tube pre-added with 25 mL 10% v/v Antibiotic- Antimycotic and 10% v/v Gentamicin in DPBS (-,-).
  • the tube was incubated at 28 °C, 29 °C, 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, 36 °C, 37 °C or 38 °C, 4% , 5% or 6% C0 2 for 2-16 hours, preferably at 37 °C, 5% C0 2 for 2 hours.
  • the foreskin was cut into small pieces (2-5mm) and transferred to a fresh 50 mL polypropylene conical centrifuge tubes containing 20 mL of 3% w/v collagenase type IV dissolved in DMEM-KO.
  • the sample was allowed to be partially digested at around 28 °C , 29 °C , 30 °C , 31 °C , 32 °C, 33 °C, 34 °C,35 °C, 36 °C, 37 °C or 38 °C, 4% , 5% or 6% C0 2 , preferably at 37 °C, 5% C0 2 for 4 to 10 hours.
  • Trypsin-EDTA 400 pL of 0.25% Trypsin-EDTA was added into the partially digested sample and incubated at 28 °C, 29 °C, 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, 36 °C, 37 °C or 38 °C, 4%, 5% or 6% C0 2 , preferably at 37 °C, 5 % C0 2 for 5 minutes to detach cells from the tissue.
  • the semi-transparent foreskin tissue fragments were transferred into a 60 mm cell culture plate using sterile tissue forceps. This process was repeated until all foreskin fragments were transferred.
  • the tissue fragments were left to air dry for 15 minutes in the biosafety cabinet. This step is to ensure the tissues adhere to the culture plate surface.
  • FSF culture media Upon adherence of the tissue fragments onto the culture plate, 5-7 mL of FSF culture media was gently added without disrupting the attached tissues.
  • the FSF culture media provides sufficient nutrients and optimal environment for the survival of the tissues.
  • the culture plates were incubated in a C0 2 incubator or low oxygen hypoxia incubator at 28 °C, 29 °C, 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, 36 °C, 37 °C or 38 °C, 4%, 5% or 6% C0 2 , preferably at 37°C, 5% C0 2 for 24 hours. 16. After 24 hours incubation, two-third of the medium was replaced with fresh FSF culture medium for continuous supply of nutrients for cell growth and also to remove dead or apoptotic cells from the culture. Avoid disrupting the adhered tissues.
  • the culture was observed using an inverted microscope every day and two-third of medium was replaced every 2 to 4 days until the cells reach 80% confluency.
  • the conditioned medium was collected and kept in a sterile bottle.
  • the conditioned medium was stored in -20 °C, -30 °C, -40 °C, -50 °C, -60 °C, - 70°C or -80°C.
  • the cell suspension was collected in 50 mL tube. 12. DPBS was added again accordingly to wash the flask surface. The suspension was collected in the same 50 mL tube.
  • the tube was centrifuge at 1,000 x g, 1,100 x g, 1,200 x g, 1,300 x g or 1,400 x g for 4-8 minutes, preferably at 1,200 x g for 6 minutes.
  • Appropriate volume of cell suspension to be seeded on cell culture flask was calculated according to the required seeding density (1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800 1,900 or 2,000 cells per cm 2 , preferably 1,500 cells per cm 2 ) and surface area of the flask.
  • the remaining cell suspension was centrifuged at 1,000 x g, 1,100 x g, 1,200 x g, 1,300 x g, or 1,400 x g for 4-8 minutes, preferably at 1,200 x g for 6 minutes.
  • Freezing medium was prepared by mixing 90% v/v fetal bovine serum (FBS) with 10% v/v dimethyl sulfoxide (DMSO). Make sure the light of the biosafety cabinet is turned off as DMSO is very sensitive to light. Keep the freezing media in the refrigerator as DMSO is toxic to the cells at room temperature.
  • FBS v/v fetal bovine serum
  • DMSO dimethyl sulfoxide
  • the cell pellet was resuspended with freezing medium (5 million of cells in 1.5 mL freezing media per cryovial). Make sure that the freezing medium is chill and cool during the whole process. Avoid any exposure of light.
  • Mr. Frosty was transferred into freezer at -70 °C, -75 °C, -80 °C or -85 °C.
  • Conditioned medium was taken out from the fridge or freezer.
  • the conditioned medium was thawed and sterile filtered using at 0.2 pm filter unit.
  • the vials were frozen at -20°C, -30 °C, -40 °C, -50°C, -60 °C, -70 °C or -80 °C.
  • the frozen growth factors were freeze dried for 24 hours to 48 hours.
  • the vials were capped with stopper and aluminum cap.
  • the growth factors were kept in - 70 °C, -75 °C, -80 °C or -85 °C for long storage.
  • Table 5 below provides a cytokine analysis on a human stem cell conditioned medium as described herein.
  • the collected stem cell conditioned medium was filtered using Akta flux machine (GE healthcare). 2. The sample was first filtered through a 0.1 pm hollow fiber. The permeate was collected.
  • the collected permeate was filtered through a 300 kDa hollow fiber.
  • the permeate was collected and labelled as“sample 4480A”.
  • the samples were frozen -20°C, -30 °C, -40 °C, -50°C, -60 °C, -70 °C or -80 °C.
  • the protein samples were reduced, alkylated and trypsin digested before being desalted.
  • Shimadzu Prominence nano HPLC system Shimadzu Prominence nano HPLC system (Shimadzu) coupled to a 5600 TripleTOF mass spectrometer (Sciex).
  • the peptides were loaded according to table 6 onto an Agilent Zorbax 300SB-C18, 3.5 pm (Agilent Technologies) and separated with a linear gradient of water/acetonitrile/0.1% formic acid (v/v).
  • Taxonomy Homo sapiens (Human)
  • Table 7 shows the identified proteins in current research Table 7
  • Exosomes were isolated from human foreskin fibroblast or fetal fibroblast. The conditioned medium from the fibroblast cell culture was collected from day 4-14 after seeding. A flow chart for the exosome purification procedure, based on differential ultracentrifugation, is shown in Figure 3. The speed and length of each centrifugation are indicated to the right of the arrows. After each of the first three centrifugations, pellets (cells, dead cells, cell debris) were discarded, and the supernatant was kept for the next step. In contrast, after the two 100,000 x g centrifugations, pellets (exosomes + contaminant proteins, exosomes) were kept, and supernatants were discarded.
  • the tubes were centrifuged at 100 x g, 200 x g, 300 x g, 400 x g or 500 x g for 5 to 15 minutes, preferably at 300 c g for 10 minutes at 4°C ( ⁇ 2°C) to remove cells.
  • Step 1-4 can be combined by centrifuging at 1,800 x g, 1,900 x g, 2, 000 x g, 2,100 x g or 2,200 x g for 18-22 minutes, preferably at 2, 000 x g for 20 minutes)
  • the tubes were centrifuged at 8,000 x g, 9,000 x g, 10,000 x g, 11,000 x g or 12,000 x g, preferably at 10,000 x g for 30 minutes at 4°C to remove cell debris.
  • the supernatant was transferred into polyallomer tubes or polycarbonate bottles appropriate for ultracentrifugation rotor to be used.
  • the tubes were centrifuged at 100,000 c g for at least 1 hour at 4 °C ( ⁇ 2 °C).
  • the pellet was resuspended in each tube with 1 mL of DPBS using a micropipettor.
  • the tube was centrifuged at 100,000 c g for at least 1 hour at 4 °C ( ⁇ 2 °C).
  • exosomes were stored at -70 °C, -75 °C, -80 °C, -85 °C or -90 °C for longer storage (avoid freeze thaw cycles).
  • the exosome was diluted in 12 mL normal saline (0.9% NaCl) in a 50 mL polypropylene conical centrifuge tube.
  • the folded capillary zeta cell was filled with the prepared exosomes by using a sterile dropper. Avoid creating bubbles inside the cell.
  • Half of the disposable sizing cuvette was filled with the prepared exosomes by using a sterile dropper.
  • the vials were frozen at -10 °C, -15 °C, -20 °C, -25 °C or -30 °C for 18-26 hours.
  • the frozen samples were freeze dried for at least 24 hours.
  • freeze-dried powder was mixed with 5mL of carrier oil or using other formulation as herein described.
  • freeze-dried powder was mixed with 5mL saline for injection or using other formulation as herein described.
  • Figure 5 illustrates the microscopic views and appearance of growth factors from SHED and WJSC mixed with different carriers, showing the microcapsulization of the growth factors and micromolecules and exosomes in the carrier oils and how their effectiveness varies depending on the oil used.
  • Table 11 shows the quantification results for protein and microRNA in different carriers after freeze-drying process.
  • Table 12 shows the quantification results for protein and microRNA in different carriers after 1 week of freeze-drying process.
  • Provitamin B5 was weighed as required and added into the phase C. The mixture was stirred manually.
  • Stem cells/ fibroblast conditioned media was weighed and added into the phase C. The mixture was stirred manually.
  • Vitamin E was weighed as required and added into the Phase D. The mixture was stirred manually.
  • Phase E preservative was weighed separately and labelled as phase E.
  • Phase F Emulsifier CP was weighed as required and labelled as phase F.
  • Phase B was added into Phase A. The mixture was stirred gently.
  • Phase C was added into the batch. The mixture was stirred gently.
  • Phase D was added into the batch. The mixture was stirred gently.
  • Phase E was added into the batch. The mixture was stirred gently.
  • Phase F was added into the batch. The mixture was stirred until homogenised.
  • the serum was homogenised using cosmetic homogenizer for 10 to 30 minutes,
  • the final product should have an appearance of semi viscous translucent white emulsion fluid with pH ranges between pH 5 - 6.
  • Niacinamide was weighed as required and added into the phase A. The mixture was stirred. PHASE B
  • Stem cells/ fibroblast conditioned media was weighed as required and labelled as Phase C.
  • Centella Asiatica extract was weighed as required and added into Phase D. The mixture was stirred.
  • Phenoxyethanol was weighed as required and labelled as Phase E.
  • Phase B was added into Phase A. The mixture was stirred gently.
  • Phase C was added into the batch. The mixture was stirred gently.
  • Phase D was added into the batch. The mixture was stirred gently.
  • Phase E was added into the batch. The mixture was stirred gently for 10 to 30 minutes, preferably 20 minutes, until homogenised.
  • the final product should have an appearance of brownish transparent fluid with a pH ranges between pH 6 - 7.
  • Stem cells/ fibroblast conditioned media was weighed as required and labelled as Phase C.
  • Provitamin B5 was weighed as required and added into the phase C. The mixture was stirred manually.
  • Phenoxyethanol was weighed as required and labelled as Phase D. MIXING
  • Phase B was added into phase A. The mixture was stirred gently.
  • Phase C was added into the batch. The mixture was stirred gently.
  • Phase D was added into the batch. The mixture was stirred gently for 10 to 30 minutes, preferably 20 minutes, until homogenised.
  • the final product should have an appearance of yellowish transparent gel with pH ranges between pH 7 - 8.
  • Facial treatment serum, facial treatment essence and NaTH plus regenerating gel were extracted from its falcon tubes using a luer lock syringe to minimize shearing of the samples, which was then connected to a blunt needle used to transfer the sample into the measurement cell. The sample was filled up to the filling height of the sample cell (approximately 1ml of sample). 3. The samples should be tested at a“fast-run” (short measurement duration) and“slow-run” (long measurement duration) to check for linear dependency on any external forces due to the centrifugation process that may affect the sample’s phase separation.
  • Table 18 shows the instability index for facial treatment serum, facial treatment essence and NaTH regenerating gel.
  • the values obtained from the stability analysis are dimensionless and has a range from 0 to 1. Zero being the most stable as the sample has not changed over the period of the measurement time and one being the most unstable as the sample has drastically changed, through phase separation, over the period of the measurement time.
  • WJSCs from passage 1 were revived and seeded into 6 well plate (TPP) (triplicates) with a seeding density of 1500 cells/cm 2 in cell culture medium. 2. One-third of conditioned medium was changed every three days.
  • TPP well plate
  • Ni is the inoculum cell number
  • N H is the cell harvest number
  • t is the time of the culture (in days)
  • Figure 6 shows the population doubling trend in WJSCs.
  • WJSCs showed a stable population doubling throughout passage 1 to passage 3 which were within the range of 2 to 3 days.
  • Cell surface marker analysis was performed using BD StemflowTM Human MSC Analysis Kit (BD Pharmigen) and analysed using BD FACSVia flow cytometer (BD Pharmigen).
  • the positive antibody cocktail consists of CD105 PerCP-CyTM5.5, CD73 APC and CD90 FITC, while the negative antibody cocktail consists of CD45, CD34, CDl lb, CD19 and HLA-DR PE.
  • a sample drop-in marker (CD44) and isotype controls were also included.
  • the cells were washed with 500 pL of DPBS and centrifuged at 300 x g for 10 minutes.
  • Cells were analyzed using BD FACSVia flow cytometer and 1 x 10 4 events/cells were acquired.
  • Figure 7 shows that immunophenotyping of stem cells derived from WJSCs were negative for CD34, CD45, CD1 lb, CD19 and HLA-DR, whereas more than 90% of cells were positive for mesenchymal stem cell markers CD73, CD90, CD105, and CD44.
  • the flasks were incubated at 28-38 °C, 4-6% C0 2 , preferably at 37 °C, 5% C0 2 for 3.5 hours and followed by six extensive washes with Knockout DMEM media.
  • WJSCs from each passage were seeded into 96 well plates (TPP) for XTT analysis with a seeding density of 15000 cells/cm 2 in cell culture media.
  • PBMCs peripheral blood mononuclear cells
  • the diluted blood was slowly dispensed at an angle into centrifuge tube filled with Ficoll Paque (GE Heallthcare) by touching the pipette tip against the inner wall of the tube without disturbing the Ficoll layer (ratio 3:4 for Ficoll :blood).
  • Ficoll Paque GE Heallthcare
  • the tubes were centrifuged (without brake) at 300 x g, 400 x g or 500 x g for 30 to 40 minutes, preferably at 400 x g for 35 minutes.
  • the buffy coat containing PBMCs was collected and washed twice with DPBS (-,-) by centrifugation at 300 x g, 400 x g or 500 x g for 5 to 15 minutes, preferably at 400 x g for 10 minutes.
  • the PBMCs were resuspended in X-vivo media supplemented with 10 % v/v FBS and 1% 100X v/v glutamax and aliquoted into two tubes. One tube was added with 10 pg/mL of phytohaemagglutinin P (PHA-P, Sigma Aldrich) to activate the PBMC and the other tube was used to serve as control (without PHA-P).
  • PHA-P phytohaemagglutinin P
  • Activated PBMCs were co-cultured into 96 well plates pre-seeded with WJSCs from
  • passage 2 and passage 6 at ratios of 1 : 10, 1 :20 and 1 :40 (WJSCs: PBMCs).
  • the co-cultures were incubated at 28-38 °C, 4-6% C0 2 , preferably at 37 °C, 5% C0 2 and the proliferation rates were measured at 24, 48 72 and 96 hours using CyQUANTTM XTT Cell Viability Assay (Invitrogen, Thermo Fisher Scientific).
  • Figure 8 shows the proliferation activity of PBMC after co-culture with WJSC at passage 2 and passage 6 respectively at the ratio of 1 : 10, 1 :20 and 1 :40.
  • PBMCs proliferation was suppressed when co-culture with WJSCs from both passage 2 and passage 6.
  • Passage 2 WJSCs showed a better suppression of PBMC proliferation as compared to passage 6 WJSC.

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Abstract

La présente invention concerne une composition comprenant un milieu conditionné de cellules souches contenant des facteurs extracellulaires et une huile support, caractérisée en ce que les facteurs extracellulaires sont encapsulés par l'huile support. La présente invention concerne en outre des formulations pour des applications cosmétiques et thérapeutiques qui contiennent des facteurs combinatoires issus du milieu conditionné de cellules souches mésenchymateuses et de fibroblastes.
PCT/MY2019/050124 2018-12-21 2019-12-20 Milieu conditionné de cellules souches WO2020130800A1 (fr)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111748520A (zh) * 2020-06-30 2020-10-09 四川大学 牙囊干细胞外泌体、制法及应用、其组合物及制法
CN112451544A (zh) * 2020-12-08 2021-03-09 陕西中鸿科瑞再生医学研究院有限公司 用于改善薄型子宫内膜的组合物及其制备方法、应用
CN112708594A (zh) * 2021-01-26 2021-04-27 中科博生生物工程有限公司 脐带间充质干细胞复苏培养基及其制备方法和应用
CN113616676A (zh) * 2021-09-09 2021-11-09 青岛大学附属医院 脐血干细胞分泌物在促进子宫内膜细胞生长中的应用
CN114469855A (zh) * 2022-01-18 2022-05-13 吉林省真承药业有限公司 一种人脐带间充质干细胞诱导分化为成纤维细胞组合应用技术
CN115444805A (zh) * 2022-09-27 2022-12-09 广东金专生物科技有限公司 一种干细胞面部美容组合物及其应用
WO2023168006A1 (fr) * 2022-03-02 2023-09-07 CryoGen, LLC Compositions comprenant des exosomes, des facteurs de croissance et des fragments de cellules souches mésenchymateuses destinées à être utilisées dans le traitement de la peau
US11878036B2 (en) 2022-05-25 2024-01-23 Neuvian LLC Vaginal care compositions and methods of improving vaginal health

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6372494B1 (en) * 1999-05-14 2002-04-16 Advanced Tissue Sciences, Inc. Methods of making conditioned cell culture medium compositions
WO2005062791A2 (fr) * 2003-12-20 2005-07-14 Jeffrey Sebastian Utilisation de nutriants et de produits combines de cellules souches et de fibroblastes dans des compositions topiques
WO2012047733A2 (fr) * 2010-09-29 2012-04-12 Aidan Research And Consulting, Llc. Traitement de l'acné par le biais de milieux conditionnés
WO2016176514A1 (fr) * 2015-04-29 2016-11-03 Nugene, Inc. Aide au soin des brûlures, des cicatrices, et des plaies et bandages
WO2017035280A1 (fr) * 2015-08-24 2017-03-02 Nugene, Inc. Pansements et aides à la cicatrisation de lésion cutanée

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6372494B1 (en) * 1999-05-14 2002-04-16 Advanced Tissue Sciences, Inc. Methods of making conditioned cell culture medium compositions
WO2005062791A2 (fr) * 2003-12-20 2005-07-14 Jeffrey Sebastian Utilisation de nutriants et de produits combines de cellules souches et de fibroblastes dans des compositions topiques
WO2012047733A2 (fr) * 2010-09-29 2012-04-12 Aidan Research And Consulting, Llc. Traitement de l'acné par le biais de milieux conditionnés
WO2016176514A1 (fr) * 2015-04-29 2016-11-03 Nugene, Inc. Aide au soin des brûlures, des cicatrices, et des plaies et bandages
WO2017035280A1 (fr) * 2015-08-24 2017-03-02 Nugene, Inc. Pansements et aides à la cicatrisation de lésion cutanée

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
VAN DER POL E ET AL.: "Classification, Functions and Clinical Relevance of Extracellular Vesicles", PHARMACOLOGICAL REVIEWS, vol. 64, no. 3, 2012, pages 676 - 705, XP055155968, DOI: 10.1124/pr.112.005983 *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111748520A (zh) * 2020-06-30 2020-10-09 四川大学 牙囊干细胞外泌体、制法及应用、其组合物及制法
CN111748520B (zh) * 2020-06-30 2021-02-19 四川大学 牙囊干细胞外泌体、制法及应用、其组合物及制法
CN112451544A (zh) * 2020-12-08 2021-03-09 陕西中鸿科瑞再生医学研究院有限公司 用于改善薄型子宫内膜的组合物及其制备方法、应用
CN112708594A (zh) * 2021-01-26 2021-04-27 中科博生生物工程有限公司 脐带间充质干细胞复苏培养基及其制备方法和应用
CN112708594B (zh) * 2021-01-26 2022-02-25 中科博生生物工程有限公司 脐带间充质干细胞复苏培养基及其制备方法和应用
CN113616676A (zh) * 2021-09-09 2021-11-09 青岛大学附属医院 脐血干细胞分泌物在促进子宫内膜细胞生长中的应用
CN113616676B (zh) * 2021-09-09 2022-05-17 青岛大学附属医院 脐血干细胞分泌物在促进子宫内膜细胞生长中的应用
CN114469855A (zh) * 2022-01-18 2022-05-13 吉林省真承药业有限公司 一种人脐带间充质干细胞诱导分化为成纤维细胞组合应用技术
WO2023168006A1 (fr) * 2022-03-02 2023-09-07 CryoGen, LLC Compositions comprenant des exosomes, des facteurs de croissance et des fragments de cellules souches mésenchymateuses destinées à être utilisées dans le traitement de la peau
US11878036B2 (en) 2022-05-25 2024-01-23 Neuvian LLC Vaginal care compositions and methods of improving vaginal health
CN115444805A (zh) * 2022-09-27 2022-12-09 广东金专生物科技有限公司 一种干细胞面部美容组合物及其应用
CN115444805B (zh) * 2022-09-27 2023-08-22 广东金专生物科技有限公司 一种干细胞面部美容组合物及其应用

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