WO2020246781A1 - Procédé de micronisation de tissu mou vivant, et greffe particulaire de tissu mou vivant ainsi produite - Google Patents

Procédé de micronisation de tissu mou vivant, et greffe particulaire de tissu mou vivant ainsi produite Download PDF

Info

Publication number
WO2020246781A1
WO2020246781A1 PCT/KR2020/007174 KR2020007174W WO2020246781A1 WO 2020246781 A1 WO2020246781 A1 WO 2020246781A1 KR 2020007174 W KR2020007174 W KR 2020007174W WO 2020246781 A1 WO2020246781 A1 WO 2020246781A1
Authority
WO
WIPO (PCT)
Prior art keywords
soft
biological tissue
tissue
living tissue
micronizing
Prior art date
Application number
PCT/KR2020/007174
Other languages
English (en)
Korean (ko)
Inventor
임동진
Original Assignee
(주)카스 인 바이오
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by (주)카스 인 바이오 filed Critical (주)카스 인 바이오
Publication of WO2020246781A1 publication Critical patent/WO2020246781A1/fr

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • A61L27/3691—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B02—CRUSHING, PULVERISING, OR DISINTEGRATING; PREPARATORY TREATMENT OF GRAIN FOR MILLING
    • B02C—CRUSHING, PULVERISING, OR DISINTEGRATING IN GENERAL; MILLING GRAIN
    • B02C18/00—Disintegrating by knives or other cutting or tearing members which chop material into fragments
    • B02C18/06—Disintegrating by knives or other cutting or tearing members which chop material into fragments with rotating knives
    • B02C18/08—Disintegrating by knives or other cutting or tearing members which chop material into fragments with rotating knives within vertical containers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B02—CRUSHING, PULVERISING, OR DISINTEGRATING; PREPARATORY TREATMENT OF GRAIN FOR MILLING
    • B02C—CRUSHING, PULVERISING, OR DISINTEGRATING IN GENERAL; MILLING GRAIN
    • B02C18/00—Disintegrating by knives or other cutting or tearing members which chop material into fragments
    • B02C18/06—Disintegrating by knives or other cutting or tearing members which chop material into fragments with rotating knives
    • B02C18/16—Details
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/06—Flowable or injectable implant compositions

Definitions

  • the present invention crushes soft biological tissues such as placenta, anionic membrane, chorionic membrane, umbilical cord, and cartilage separated from humans or animals It relates to a method of converting, and the pulverized soft biological tissue fine particles are usefully used as a material for a biological tissue graft.
  • Various biological tissues can be isolated from humans or animals.
  • the separated biological tissues can be used as a medical material or a pharmaceutical material through a series of processes.
  • the separated biological tissue may be used as an allograft, preferably an allograft, for tissue repair and wound healing.
  • the living tissue graft can be used as an injectable graft or a patchable graft.
  • Biological tissues such as bone and cartilage can generally be obtained from a carcass.
  • biological tissues such as placenta, amniotic membrane, chorion, and umbilical cord can be obtained after natural delivery or cesarean section of a pregnant woman.
  • the placenta is an organ that connects the fetus with the mother's uterus, and supplies oxygen and various nutrients to the fetus.
  • the placenta also produces hormones necessary for the fetus and is known to synthesize various factors necessary for immunity.
  • the placenta has long been used as a graft for repairing human tissues or healing wounds.
  • the placenta is composed of an umbilical cord and an amniotic sac, and the amniotic sac is composed of an aminiotic membrane, a chorionic membrane, and a maternal decidua. Between the amnion and the chorionic membrane is an intermediate layer, called a "spongy layer".
  • the amniotic membrane is a thin membrane surrounding the fetus at the innermost part of the placenta, and the amniotic fluid is filled inside the amniotic membrane.
  • the amniotic membrane consists of a single epithelium layer and extracellular matrix (ECM), and the extracellular matrix is again reticular fibers (or basement membrane), a thick compact layer and fibroblasts. It consists of a fibroblast layer.
  • the chorionic membrane is composed of reticular fibers, a basement membrane, and a trophoblast.
  • amniotic and chorionic membranes contain various growth factors, cytokines, collagen, and cell-adhesive proteins, as well as stem cells.
  • Growth factors include, for example, epidermal growth factor (EGF), fibroblast growth factor (FGF), transforming growth factor Beta (TGF-b), nerve growth factor (nerve growth factor). factor, NGF), hepatic growth factor (HGF), and vascular endothelial growth factor (VEGF).
  • EGF epidermal growth factor
  • FGF fibroblast growth factor
  • TGF-b transforming growth factor Beta
  • nerve growth factor nerve growth factor
  • nerve growth factor nerve growth factor
  • NGF nerve growth factor
  • HGF hepatic growth factor
  • VEGF vascular endothelial growth factor
  • Cytokines are, for example, interleukin (IL)-1 ⁇ , IL-1 ⁇ , IL-6, IL-8, IL-10, metalloproteinase tissue inhibitors (TIMP)-1, TIMP-2, TIMP-3, TIMP- 4th place.
  • IL interleukin
  • TIMP-2 TIMP-2
  • TIMP-3 TIMP- 4th place.
  • Collagen is, for example, collagen type I, type III, type IV, type V, and type VII.
  • Cell-adhesive proteins are, for example, fibronectin, laminin, fibrillin, nidogen, proteoglycan, glycosaminoglycan, and the like.
  • Mesenchymal stem cells are known to exist in large amounts in the epithelial cell layer of the amnion and the feeder cell layer of the chorion.
  • tissue or membrane derived from the placenta has been used as a substrate for wound recovery such as diabetic foot ulcers.
  • a variety of placenta products are commercially available for wound treatment. For example, there are products such as:
  • AmnioBand ® Membrane (MTF Biologics, USA) is a dehydrated (dried) allograft derived from the human placenta and consists of the amniotic membrane and chorionic membrane.
  • Biovance ® (Alliqua BioMedical, USA) is a decellularized and dehydrated human amniotic membrane allograft.
  • Epifix ® (MiMedx Group, Inc., USA) is a dehydrated human amniotic/chorionic allograft (dHACM) used to treat diabetic foot ulcers.
  • EpiFix ® Micronized Amniotic Membrane is a micronized dHACM that can be applied to deep wounds of cartilage, bones and muscles.
  • Grafix ® (Osiris Therapeutics, Inc., USA) is a cryopreserved placenta-derived membrane composed of an extracellular matrix.
  • Grafts for these wound repairs generally do not need to be micronized.
  • the placenta-derived tissue graft may be administered into the human body by surgical incision or non-incision.
  • surgical incision an incision is made at the site of the procedure and the graft is inserted and then sutured. This surgical incision causes another scar.
  • Non-incisional surgery can be performed using an injection-type graft.
  • a tissue graft is injected into the human body, such as muscles, cartilage, and ligaments through a syringe.
  • the micronization of the tissue graft is essential, and the size of the microparticles is very important.
  • the fine particles of the graft should be sufficiently smaller than the inner diameter of the needle, and the larger the diameter of the needle, the more painful the patient will be, and may cause side effects such as skin injury or tissue destruction.
  • the size of the graft microparticles suitable for injection is at least 1000 ⁇ m or less, preferably 500 ⁇ m or less, more preferably 300 ⁇ m or less.
  • Placenta-derived grafts for injection that are commercially available include, for example, the following products.
  • AmnioFix ® Injectable (MiMedx Group, Inc., USA) is a micronized dHACM graft used to treat knee arthritis, orthopedic soft tissue, tendon injuries and tendonitis.
  • the product is a dehydrated powder formulation of an allogeneic tissue graft, and the shelf life is at least 5 years at room temperature.
  • ReNu ® and NuCel ® are cryopreserved bioactive suspension grafts containing amniotic membrane and amniotic fluid.
  • the product is used to treat degenerative diseases such as knee osteoarthritis, joint and tendon injuries.
  • Clarix ® Flo and Clarix ® Injectable are lyophilized micro human placenta allografts consisting of an amniotic membrane and umbilical cord.
  • Soft biological tissues such as placenta, amnion, chorion, umbilical cord, and cartilage must be micronized in order to be used as an injection.
  • U.S. Patent No. 9,943,551 discloses a method of pulverizing placental tissue, amnion tissue, and chorionic tissue to a microscopic size in order to make an injection composition, and the pulverizer discloses a Retsch TM MM400 ball mill and a Retsch TM Cryomill.
  • a sample and a milling ball are placed in a milling cup, the cover of the milling cup is closed, and the milling cup is inserted into a cup holder.
  • the milling cup vibrates (reciprocates) at high speed, and the sample is effectively crushed by a strong impact due to the inertia of the ball.
  • Soft biological tissues such as amnion and chorion must be almost completely dehydrated (dried) before performing a ball mill. Otherwise, it will be barked rather than crushed and the desired shape of the fine particles cannot be obtained. Since dehydration refers to a non-viable environment, cells in living tissues are destroyed or killed.
  • FIG. 2 shows a representative model of cryomill Retsch TM Cryomill.
  • the cryomyl is a structure in which a liquid nitrogen circulation system is installed in a conventional ball mill (vibration mill). Liquid nitrogen is supplied to the outside of the milling cup, and the sample is rapidly frozen and hardened for grinding. Cryomyl quickly cools the sample to -196°C for effective pulverization, so cells in living tissues, particularly stem cells, are almost killed.
  • cryomyl or ball mill is useful for micronizing soft biological tissues, but since it is crushed like a large mortar by ball strike, there is a fatal problem in cell survival of micronized biological tissues.
  • An object of the present invention is to provide a micro-pulverization method that minimizes cell destruction of living tissue.
  • Another object of the present invention is to provide a method for preparing a biological tissue graft having a high content of active ingredients per unit volume.
  • Another object of the present invention is to provide a method for pulverizing biological tissues, which has a simpler pulverization method, high production time efficiency, and low production cost compared to the prior art.
  • the present invention comprises the steps of slicing soft living tissue isolated from humans or animals using a cutting tool; And it provides a method for micronizing a soft biological tissue comprising the step of pulverizing the shredded soft biological tissue together with a solid biocompatible material in a rotor mill equipped with a sieve (sieve).
  • the biocompatible material may be a biocompatible polymer or sugar.
  • the biocompatible polymer is not limited, but serum albumin, hyaluronic acid, starch, gelatin, chitosan, collagen, alginic acid, pectin, carrageenan, chondroitin (sulfate), dextran (sulfate), Polylysine, carboxymethyl titine, fibrin, agarose, pullulan, cellulose, polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), polyvinyl alcohol (PVA), hydroxypropylcellulose (HPC), Hydroxyethylcellulose (HEC), hydroxypropylmethylcellulose (HPMC), sodium carboxymethylcellulose, polyalcohol, gum arabic, alginate, cyclodextrin, dextrin, palatinite, polylactic acid, polyglycolic acid (polyglycolic acid), polyethylene oxide, polyacrylic acid, polyacrylamide, polymethacrylic acid, and may include one or more polymer materials selected from the group consisting of polymaleic
  • the saccharide is not limited, but the group consisting of glucose, fructose, starch, trehalose, glucose, maltose, lactose, lactulose, fructose, turanose, melitose, melesitos, dextran, sorbitol, xylitol It may contain one or more saccharides selected from.
  • the biocompatible material may be serum albumin.
  • the biocompatible material may further include an active ingredient required for survival or growth of cells.
  • the active ingredients required for survival or growth of the cells are DMEM (Dulbecco's Modified Eagle Media), MEM (Minimum Essential Media), RPMI 1640, Iscove's Modified Dulbecco's Media, Defined Keratinocyte-SFM (without BPE), Keratinocyte-D. -MEM, AmnioMAX-II Complete Culture media, AmnioMAX-C100 Complete Culture media, Plasmalyate-148 and Plasmalyte-A may be selected from the group consisting of.
  • DMEM Dulbecco's Modified Eagle Media
  • MEM Minimum Essential Media
  • RPMI 1640 Iscove's Modified Dulbecco's Media
  • Keratinocyte-SFM without BPE
  • Keratinocyte-D. -MEM Keratinocyte-D. -MEM
  • AmnioMAX-II Complete Culture media AmnioMAX-C100 Complete Culture media
  • the soft biological tissue of the present invention may be selected from the group consisting of amniotic membrane, chorionic membrane, placenta, umbilical cord, and cartilage.
  • the average diameter of the pulverized soft biological tissue may be 500 ⁇ m or less.
  • the rotor mill may be provided with a plurality of blades (multi-tooth, multi-blade), and may include a rotating circular rotor and a sieve mounted on the outside thereof.
  • the present invention provides a biological tissue fine particle graft comprising the prepared biological tissue fine particles.
  • the biological tissue microparticle graft may be obtained by freezing micronized soft biological tissue.
  • the biological tissue particulate graft may be used as an injection after freezing and thawing.
  • the biological tissue pulverized by the method of the present invention has significantly higher cell viability than conventional ball mill and cryomill pulverization, so that a biological tissue graft can be provided.
  • the pulverization method according to the present invention has a low cost and very high production efficiency compared to the prior art.
  • FIG. 3A is a diagram showing a general configuration of a laboratory rotor mill
  • FIG. 3B is a diagram showing the configuration of a rotor mill separated.
  • FIG 4 (a) is a phase diagram showing the gelation area according to the concentration and temperature of serum albumin used as a grinding aid, (b) is a gelation form of 20% (w/v) bovine serum-derived albumin and human serum albumin It is a picture showing.
  • Figure 5 shows the gelled form of albumin and cell culture medium mixture
  • A (Figure 5A) is essential amino acids (AA)
  • B ( Figure 5B) is vitamins (VITA)
  • C ( Figure 5C) is non-essential Amino acids (NAA) are added.
  • A is essential amino acids (AA)
  • B is vitamins (VITA)
  • C is a non-essential amino acid (NAA) is added, and glutathione in these mixtures The concentration is 10mM.
  • A is a grinding aid prepared with only 20% (w/v) albumin
  • B is an essential amino acid (5 ⁇ ), a non-essential amino acid (5 ⁇ ), and a vitamin (1 ⁇ ). It shows a grinding aid prepared with 20% (w/v) albumin contained.
  • FIG. 8A is a micrograph of an amniotic membrane tissue pulverized with a conventional cryomill
  • B is a microscopic picture of amnion tissue pulverized according to Example 4.
  • ECM 10 is a scanning electron microscope image showing the appearance of the extracellular matrix (ECM) of the amniotic membrane tissue pulverized according to Example 4 (magnification: 10000 times).
  • FIG. 11 is a photograph showing the results of treatment with the Alamar Blue reagent of the amnion graft according to the present invention.
  • FIG. 12 is a graph showing the results of measuring cell metabolic activity of the amniotic membrane graft according to the present invention using a fluorescence photometer.
  • FIG. 13 is a graph showing the effect of promoting the growth of human fibroblasts of the amniotic membrane graft prepared according to the present invention.
  • living tissue refers to a viable tissue including living cells or viable cells.
  • soft in "soft biological tissue” used in the present invention is a soft tissue or material isolated from humans or animals, and includes, for example, placenta, amniotic membrane, chorion, umbilical cord, cartilage, skin, and ligaments.
  • fine-particleization means pulverizing a soft biological tissue to a size of 1000 microns or less.
  • biocompatible material used in the present invention means a material that is not harmful to cells or tissues constituting a cartilage tissue graft.
  • solid form is used as a meaning including a hard form as well as a gel form.
  • the "pulverization agent or grinding agent" used in the present invention is a material added to effectively grind soft biological tissues in a rotor mill.
  • the "purging agent" used in the present invention is a material that pushes the soft biological tissue pulverized in the rotor mill to effectively exit the pores of the ring sieve.
  • the present inventors have made various attempts to effectively micronize soft biological tissues such as amniotic membrane, chorionic membrane, placenta, and umbilical cord while improving the cell viability of micronized soft biological tissue.
  • the inventors of the present invention confirmed that when crushing conventional ball mills, vibration mills, cryo mills used for crushing the amniotic membrane and chorionic membranes, cells are destroyed as these living tissues are previously dehydrated (dried) or rapidly frozen, and almost all cells are killed after fine particles I did.
  • the present inventors pulverized the soft biological tissue by putting a solid biocompatible material together as a pulverization agent or a purging agent, and surprisingly, the pulverization was smoothly performed, and the crushed tissue was smooth. It was confirmed that it passed through a sieve. In addition, it was found that the cell viability of the pulverized tissue is increased when a pulverization aid is used by adding components capable of inducing cell protection and activity to such a biocompatible material.
  • the biocompatible material used as a grinding aid or purging agent in the present invention is not limited, but, for example, a biocompatible low molecule, a biocompatible polymer may be used, and a natural polymer, a synthetic polymer, or a recombinant polymer may be used.
  • the placenta is obtained after spontaneous delivery or cesarean section surgery with the informed consent of the pregnant woman.
  • pregnant women are pre-tested for HIV-1, HIV-2, HTLV-1, hepatitis B virus and syphilis virus using conventional serological tests. .
  • the above test items are exemplary, and more serum tests may be performed if necessary.
  • the placenta can be used for the graft of the present invention only if the pregnant woman's blood is serologically negative.
  • Placenta meeting the above criteria is placed in a sterile plastic bag, stored in an ice bucket, and transferred to the laboratory as soon as possible. The following procedure is performed in aseptic conditions routinely used for tissue culture. The sterile plastic bag containing the placenta is checked and stored in the refrigerator until ready for further processing.
  • Blood and clots remaining in the placenta can be removed using saline.
  • the amniotic and chorionic membranes from the placenta can be carefully separated by hand.
  • the separated amniotic membrane and chorionic membrane may be pulverized individually or in combination with a rotor mill to be described later.
  • the layer of epithelial cells of the amnion may be micronized without being removed.
  • the epithelial cell layer of the amnion can be physically or chemically removed by known techniques.
  • the epithelial cell layer may be removed using a cell scraper, or nonionic surfactants such as Triton X-100, Triton X-114, NP-40, Brij-40, and Tween-80, ionic surfactants, or nucleases. Can be removed.
  • the amniotic membrane is washed with physiological saline or PBS added with antibiotics.
  • Streptomycin Streptomycin
  • gentamicin Gentamicin
  • penicillin polymycin B
  • Polymixin B Polymixin B
  • bacitracin Bacitracin
  • a mixture thereof, etc. are used as antibiotics, but all antibiotics known in the art can be used. have.
  • the soft biological tissue is cut (minced) into small size using a cutting tool.
  • the cutting tool is irrelevant as long as it can cut living tissue, but a knife or scissors can be used mainly.
  • a knife for slicing living tissue for example, a rotating knife (rolling knife), a round knife, a straight knife, a mincer knife, etc. can be used, and these knives are arranged at regular intervals.
  • a knife may be used.
  • the living tissue may be shredded by an electric grinder equipped with a knife blade such as a household electric mixer.
  • the soft biological tissue can be shredded in a grid pattern.
  • the soft living tissue can be shredded into amorphous shape using a cutting tool.
  • the size of the fragmented biological tissue is not limited, but may be 1 to 10 mm in diameter, 2 to 9 mm, 3 to 8 mm, 4 to 7 mm, and 5 to 6 mm.
  • the rotor mill grinder is a device that grinds materials with centrifugal force and shear force generated by the rotation of the rotor.
  • FIG. 3A is a diagram showing a general configuration of a laboratory rotor mill
  • FIG. 3B is a diagram showing the configuration of a rotor mill separated.
  • the rotor mill includes a body 1 and a cover 2.
  • a rotor 3 is provided at a central position of the main body 2, and a circular body 4 surrounds the rotor 3.
  • the rotor 3 is rotated at high speed by a motor, and the circular body 4 is fixed to the outside of the rotor 3.
  • the rotor 3 may be a rotor having a multi-tooth 5.
  • the blades 5 of the rotor may be arranged vertically on the circumference of the rotor at regular intervals from each other.
  • the blade 5 of the rotor may have a triangular shape of a stainless steel bar or a titanium bar.
  • the number of blades 5 of the rotor may be 4, 6, 8, 12, or 24, but is not limited thereto. In general, as the number of rotor blades 5 increases, the size of the pulverized fine particles decreases.
  • a number of micropores are formed on the surface of the circular body 4.
  • the shape of the micropores may be a round hole or a trapezoidal hole, but is not limited thereto.
  • the size of the micropores is not limited, but may be less than 1.00 mm, less than 0.75 mm, less than 0.50 mm, or less than 0.25 mm.
  • the size of the micropores of the prototype may be appropriately selected according to the size of the fine particles of the soft living tissue.
  • the shredded soft biological tissue may be pre-positioned on the center of the rotor.
  • the shredded soft biological tissue may be supplied to the center of the rotor through the inlet 7 of the rotor mill.
  • the soft biological tissue shredded by the centrifugal force generated by the high-speed rotation of the rotor is moved toward the circular body (4), and is torn by the shear force between the blade (5) and the circular body (4) of the rotor. It is ground, ground and micronized.
  • the micronized soft biological tissue passes through the micropores formed in the circular body 4 and is collected in an external collection container.
  • Rotor mills are for example Retsch ® ZM 200 (Retsch GmbH, Germany), Retsch ® ZM 100 (Retsch GmbH, Germany), Retsch ® ZM 1000 (Retsch GmbH, Germany), Retsch ® TWISTER (Retsch GmbH, Germany), Retsch ® SR300 (Retsch GmbH, Germany), Fritsch ® Pulverisette14 (Fritsch GmbH, Germany), Glatt Rotor Sieve GSE may be used, but are not limited thereto.
  • the grinding aid or purging agent may be a solid or gel biocompatible material.
  • the solid form may be a hard formulation, or may be a soft formulation like jelly.
  • the biocompatible material may be prepared by itself or in a solid form or a gel form by mixing a solution such as water or a medium.
  • the biocompatible material may be manufactured by cutting with a knife or making a tablet in a mold. If necessary, a biocompatible adhesive may be added to the biocompatible material.
  • the biocompatible material may include ingredients that are not harmful in the human body.
  • the biocompatible material is not limited, but may be a component used for medical purposes, a component used as an excipient for a drug, or a component used for a medical device.
  • the biocompatible material may be a low-molecular material or a high-molecular material as long as it is maintained in a solid form or a gel form.
  • the polymer material natural polymers, recombinant polymers, and synthetic polymers can be used.
  • the biocompatible material is not limited, but serum albumin, hyaluronic acid, starch, gelatin, chitosan, collagen, alginic acid, pectin, carrageenan, chondroitin (sulfate), dextran (Sulfate), polylysine, carboxymethyl titine, fibrin, agarose, pullulan, cellulose, polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), polyvinyl alcohol (PVA), hydroxypropylcellulose (HPC), hydroxyethylcellulose (HEC), hydroxypropylmethylcellulose (HPMC), sodium carboxymethylcellulose, polyalcohol, gum arabic, alginate, cyclodextrin, dextrin, palatinite, polylactic acid , Polyglycolic acid, polyethylene oxide, polyacrylic acid, polyacrylamide, polymethacrylic acid, and polymeric materials such as polymaleic acid may be used.
  • PVP polyvinylpyrrolidone
  • the biocompatible material is not limited, but glucose, fructose, starch, trehalose, glucose, maltose, lactose, lactulose, fructose, turanose, melitose, melesitos, dextran, sorbitol, It may be a sugar such as xylitol.
  • the starch used in the grinding aid may be pharmaceutical starch or pregelatinized starch, and may be included in whole or in part.
  • PGS grade pregelatinized starch such as Cargill's C ⁇ PharmGelTM can be used.
  • the biocompatible material may be water-soluble. At this time, after grinding with a rotor mill, the biocompatible material is dissolved in an aqueous solution, so it can be easily removed through a filter.
  • the biocompatible polymer may be a biodegradable polymer.
  • the grinding aid or purging agent may contain an active ingredient required for survival or growth of cells.
  • the active ingredient may be a culture medium for cell growth.
  • the culture medium is not limited, but DMEM (Dulbecco's Modified Eagle Media), MEM (Minimum Essential Media), RPMI 1640, IMDM (Iscove's Modified Dulbecco's Media), Defined Keratinocyte-SFM (without BPE), Keratinocyte-SFN (with BPE) ), KnockOut D-MEM, AmnioMAX-II Complete Medium and AmnioMAX-C100 Complete Medium, Plasmalyate-148, Plasmalyte-A, and the like may be used.
  • the active ingredient may be amniotic fluid or amniotic fluid-derived ingredients.
  • the biocompatible material may be in a solid form or a gel form, and the size is not limited, but the average diameter is 1 to 8 mm, preferably 2 to 7 mm, more preferably 3 to 6 mm. It may be, even more preferably 4 ⁇ 5mm.
  • the solid biocompatible material may be prepared by using a pill tablet. If necessary, purified water may be added.
  • the biocompatible material when the biocompatible material is a sugar such as glucose or sucrose, it may be mixed with purified water and then put in a mold and dried.
  • the solid biocompatible material may be prepared by crosslinking by adding a crosslinking agent in a liquid phase.
  • a crosslinking agent glutaraldehyde may be used, but the present invention is not limited thereto, and a known crosslinking agent may be used.
  • the solid biocompatible material may be dissolved in an aqueous solution, mixed with an excipient such as starch, and dried.
  • the grinding aid or purging agent is divided into roles, and the grinding aid and purging agent may be the same. That is, the grinding aid can be a purging agent, and the purging agent can be a grinding aid. In one aspect, the purging agent may be a biocompatible material described in the aforementioned grinding aid.
  • the grinding aid or purging agent When the rotor mill is operated, the grinding aid or purging agent is crushed between the rotor blade and the circular sieve by centrifugal and shear forces.
  • a grinding aid or purging agent not only helps the pulverization of soft living tissues, but also serves to push the pulverized living tissues out of the prototype body (purging agent).
  • the grinding aid serves to buffer the impact generated by the shear of the rotor mill to prevent or reduce damage or destruction of cells including tissues.
  • the operation sequence of the rotor mill is as follows.
  • the cover 2 of the rotor mill is opened, and a labyrinth disc 8 is inserted on the body of the rotor mill.
  • a rotor (3) equipped with a multi-blade is mounted on the central axis of the motor, a collection container (cassette) (6) is mounted, and then a circular sieve (4) is mounted.
  • the shredded soft living tissue is placed in the center of the rotor (3), and the grinding aid is placed in a position slightly away from the center of the rotor. Operate after closing the cover of the rotor mill.
  • the rotational speed and operating time of the rotor mill can be preset.
  • the rotor mill is not limited, but rotates at high speeds of 1,000-18,000 rpm. Meanwhile, when a separate purging agent is injected through the rotor mill inlet during operation of the rotor mill, the pulverized soft biological tissue is completely pushed out of the circular body.
  • the pulverized biological tissue remaining in the prototype can be collected using a washing solution or a culture medium, and the pulverized biological tissue can then be obtained by filtration or centrifugation.
  • the obtained biological tissue microparticles can be stabilized in a culture medium or a stabilizing solution.
  • antioxidants such as N-acetyl cysteine (NAC) may be further added to the culture medium or stabilization solution.
  • the micronized soft biological tissue of the present invention may include living cells, for example, mesenchymal stem cells, epithelial cells, amnion-derived fibroblasts, and the like.
  • the micronized soft biological tissue including the living cells may be cryopreserved.
  • Cryoprotective agents can be added to prevent cell damage while freezing the cells.
  • cryoprotectants There are two types of cryoprotectants: cell-permeable cryoprotectants and non-permeable cryoprotectants.
  • Cell-permeable cryoprotectants are low-molecular substances capable of penetrating cell membranes, and play a role of inhibiting the formation of ice cubes upon freezing by substituting water inside cells through the cell membrane.
  • the impermeable cryoprotectant is mainly a polymer material, and serves to inhibit the formation of ice crystals outside the cell.
  • DMSO dimethyl sulfoxide
  • EG ethylene glycol
  • Gly glycerol
  • MeOH methanol
  • PG propylene glycol
  • Non-permeable cryoprotectants include lactose, dextran 40, dextran 70, fructooligosaccharide, isomaltooligosaccharide, galactooligosaccharide, and penta.
  • Isomaltose pentaisomaltose
  • trehalose trehalose
  • sucrose sucrose
  • raffinose raffinose
  • d-mannitol mannito
  • the cell permeable cryoprotectant and the impermeable cryoprotectant may be used alone or in combination.
  • concentration of the cryoprotectant is not limited, but may be 5 to 40% (v/v).
  • a mixture of 3-5% DMSO and 9% dextran 70 may be used.
  • the cryopreservation method of living tissue can be performed according to a cryopreservation protocol known in the art.
  • the soft biological tissue microparticle graft of the present invention may be used in the form of an injection after melting, and is mainly used for orthopedic treatment such as knee osteoarthritis, soft tissue damage, ligament damage, and tendon damage. I can.
  • Bovine serum albumin BSA
  • HSA human serum albumin
  • (a) is a phase diagram showing the gelation area according to the concentration and temperature of serum albumin used as a grinding aid, and (b) is a gelation of 20% (w/v) bovine serum-derived albumin and human serum albumin. Show the form.
  • Example 2 Gelation of albumin + cell culture medium mixture (preparation of a grinding aid)
  • the albumin solution and the cell culture medium were mixed to perform gelation of albumin.
  • Cell culture medium is MEM amino acid solution (50 ⁇ : 50 times concentrate) containing essential amino acids (L-Arginine, L-Cystine, L-Histidine, L-Isoleucine), and non-essential amino acids (L-Alanine, L-Asparagine).
  • L-Aspartic acid containing MEM non-essential amino acid solution (100 ⁇ : 100 times concentrate)
  • vitamins containing MEM vitamin solution 100 ⁇ : 100 times concentrate
  • the albumin concentration was adjusted to a concentration of 20% (w/v) with respect to the total mixture, and the concentration of amino acids and vitamins was diluted with respect to the total mixture. For example, if 400 ⁇ l of 25% (w/v) albumin solution and 100 ⁇ l of amino acids (100 ⁇ ) are mixed, a total of 500 ⁇ l is obtained, while albumin is diluted to 20% (w/v) and amino acids are 20 times (20). ⁇ ).
  • Figure 5 shows the gelled form of albumin and cell culture medium mixture
  • A (Figure 5A) is essential amino acids (AA)
  • B ( Figure 5B) is vitamins (VITA)
  • C ( Figure 5C) is non-essential Amino acids (NAA) are added.
  • both albumin and cell culture medium mixtures were gelled, and in the case of non-essential amino acids, when 10 times or more was added, a translucent or milky albumin gel was formed.
  • a 20% (w/v) human serum albumin solution, pregelatinized starch, and the cell culture medium mentioned in Example 2 were mixed to prepare a partial starch paste.
  • Figure 6 shows the form of the albumin starch paste
  • A (Figure 6A) is essential amino acids (AA)
  • B ( Figure 6B) is vitamins (VITA)
  • C ( Figure 6C) is non-essential amino acids (NAA) Is added.
  • albumin and albumin starch paste were all prepared in a milky gel form.
  • Triton X was treated, and then the epithelial cell layer of the amniotic membrane was removed using a cell scraper.
  • amniotic membrane was washed with PBS or physiological saline to which 1% streptomycin/penicillin antibiotic was added, and then stabilized in DMEM medium.
  • the amnion was shredded into a size of about 4mm ⁇ 4mm using a rolling multi-blade.
  • the albumin gel prepared in the same manner as in Examples 1 and 2 was cut into cubes and used as a grinding aid.
  • A is a grinding aid prepared with only 20% (w/v) albumin
  • B is an essential amino acid (5 ⁇ ), a non-essential amino acid (5 ⁇ ), and a vitamin (1 ⁇ ). It shows a grinding aid prepared with 20% (w/v) albumin contained.
  • a 6-bladed rotor and a ring sieve having a diameter of 0.25 mm were mounted on a rotor mill (retsch zm 200).
  • the pulverized amnion adhered to the collection container and sieve of the rotor mill was washed with DMEM medium to collect the pulverized amnion, and stabilized at room temperature for 1 hour.
  • Example 4 by observing the tissue with the amniotic membrane and the amniotic membrane tissue ground to a conventional keurioh mill (Retsch Cryomill TM) milling according to the microscope naeteotda receive the results in Figs. 8 and 9 (Scale Bar: 200 ⁇ m) .
  • FIG. 8A is a micrograph of a conventional amnion tissue pulverized with a cryomill
  • B is a micrograph of a pulverized amnion tissue according to Example 1.
  • FIG. 8B it can be seen that the cells in the amniotic membrane tissue crushed with conventional cryomyl are all destroyed, whereas the amniotic membrane tissue according to Example 4 survives.
  • FIG. 9 is another micrograph of the amniotic membrane tissue pulverized according to Example 4. As shown in Figure 9, it can be seen that the size of the micronized amniotic membrane tissue according to the present invention is about 500 ⁇ m or less.
  • ECM extracellular matrix
  • Each amniotic membrane graft prepared according to the present invention was branched into a 24-well plate, incubated in 20% serum high-concentration glucose DMEM medium (1 ml) for 12 hours, and a recovery period was held for 12 hours.
  • FIG. 11 shows Alamar Blue reagent-treated amniotic membrane grafts, where "Fresh Amniotic Tissue” represents unground amniotic membrane grafts, and "Processed Amniotic Tissues” represents amniotic membrane grafts pulverized according to the present invention.
  • the fluorescence intensity of the well plate was measured using a fluorescence photometer to measure cell metabolic activity, and the results are shown in FIG. 12 (Ex: 560 nm; Em: 590 nm).
  • the amniotic membrane graft according to the present invention has a lower cell metabolic activity compared to the uncrushed amnion tissue, but it means that the cell survival is possible even after pulverization.
  • a grinding aid made of 20% (w/v) albumin containing essential amino acids (5 ⁇ ), non-essential amino acids (5 ⁇ ) and vitamins (1 ⁇ ) has the highest cellular metabolism activity compared to other grinding aids. Can be seen.
  • a mixed solution of the amniotic membrane graft and serum-free medium 1:1 (w/w) according to the present invention was prepared, a serum-free medium was prepared as a negative control, and a 10% FBS medium was prepared as a positive control.
  • Human fibroblasts (Detroit 562) were mixed with 1 ml of serum-free medium, and 3 wells were inoculated into well plates of a total of 3 groups at 5 ⁇ 10 5 per well, and the cells were attached for 24 hours. After mounting a culture plate insert having a 3 ⁇ m pore (cell culture plate insert), the amnion graft mixture solution, the negative control group, and the positive control group according to the present invention were respectively added to the culture plate inserts and cultured for 24 hours.
  • the MTT reagent was dissolved in PBS (phosphate-buffered saline) at a concentration of 5 mg/mL, treated with 100 ⁇ l each in a cultured 24-well plate, and stored at 37°C for 1 hour, and the amount of MTT formazan produced at 540 nm absorbance was measured. By quantification, the cell activity of all cells was analyzed.
  • PBS phosphate-buffered saline
  • FIG. 13 is a graph showing the effect of promoting the growth of human fibroblasts of the amniotic membrane graft prepared according to the present invention.
  • amniotic membrane graft Provided Amniotic Tissues
  • the amniotic membrane graft showed a high MTT activity (activity) compared to the negative and positive control groups, which included an active ingredient that helps cell growth in the amniotic membrane graft according to the present invention. It means being
  • the present invention crushes soft biological tissues such as placenta, anionic membrane, chorionic membrane, umbilical cord, and cartilage separated from humans or animals It relates to a method of converting, and the pulverized soft biological tissue fine particles are usefully used as a material for a biological tissue graft.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biomedical Technology (AREA)
  • Transplantation (AREA)
  • General Health & Medical Sciences (AREA)
  • Botany (AREA)
  • Dermatology (AREA)
  • Medicinal Chemistry (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Molecular Biology (AREA)
  • Food Science & Technology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Zoology (AREA)
  • Materials For Medical Uses (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Cosmetics (AREA)

Abstract

La présente invention concerne un procédé de micronisation de tissu mou vivant, le procédé comprenant : une étape dans laquelle un tissu vivant mou isolé à partir d'un être humain ou d'un animal est tranché à l'aide d'un outil de découpe ; et une étape de broyage du tissu mou vivant tranché avec un matériau biocompatible solide dans un broyeur à rotor équipé d'un tamis.
PCT/KR2020/007174 2019-06-02 2020-06-02 Procédé de micronisation de tissu mou vivant, et greffe particulaire de tissu mou vivant ainsi produite WO2020246781A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR10-2019-0065035 2019-06-02
KR20190065035 2019-06-02

Publications (1)

Publication Number Publication Date
WO2020246781A1 true WO2020246781A1 (fr) 2020-12-10

Family

ID=73653007

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2020/007174 WO2020246781A1 (fr) 2019-06-02 2020-06-02 Procédé de micronisation de tissu mou vivant, et greffe particulaire de tissu mou vivant ainsi produite

Country Status (2)

Country Link
KR (1) KR102487903B1 (fr)
WO (1) WO2020246781A1 (fr)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006043578A (ja) * 2004-08-04 2006-02-16 Nagao System:Kk 遊星ボールミル
US20150132851A1 (en) * 2011-11-08 2015-05-14 Auxocell Laboratories, Inc. Systems and methods for processing cells
JP2017529152A (ja) * 2014-08-28 2017-10-05 ミメディクス グループ インコーポレイテッド コラーゲンで強化した組織移植片
JP2018503103A (ja) * 2014-12-15 2018-02-01 ヒューマン ブレイン ウェイブ エス．アール．エル． 生体物質の分解装置および対応する製造方法、並びに、細胞懸濁液および組織微小移植片の調製方法
WO2018064975A1 (fr) * 2016-10-04 2018-04-12 Transwell Biotech Co., Ltd. Compositions et méthodes pour déterminer une viabilité cellulaire

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE2947908A1 (de) * 1979-11-28 1981-06-04 Frigocyt Labor Dr. Gosch & Sohn GmbH, 2000 Hamburg Verfahren zur herstellung von tiefgefrorenen, keimfreien und zoonosefreien zellpraeparaten aus rinderfetus o.dgl.
KR20240042159A (ko) * 2011-02-14 2024-04-01 미메딕스 그룹 인크. 마이크로화된 태반 조직 조성물 및 이의 제조 및 사용 방법
US20220213430A1 (en) * 2019-04-24 2022-07-07 Viadigm Llc Methods of tissue processing

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006043578A (ja) * 2004-08-04 2006-02-16 Nagao System:Kk 遊星ボールミル
US20150132851A1 (en) * 2011-11-08 2015-05-14 Auxocell Laboratories, Inc. Systems and methods for processing cells
JP2017529152A (ja) * 2014-08-28 2017-10-05 ミメディクス グループ インコーポレイテッド コラーゲンで強化した組織移植片
JP2018503103A (ja) * 2014-12-15 2018-02-01 ヒューマン ブレイン ウェイブ エス．アール．エル． 生体物質の分解装置および対応する製造方法、並びに、細胞懸濁液および組織微小移植片の調製方法
WO2018064975A1 (fr) * 2016-10-04 2018-04-12 Transwell Biotech Co., Ltd. Compositions et méthodes pour déterminer une viabilité cellulaire

Also Published As

Publication number Publication date
KR102487903B1 (ko) 2023-01-12
KR20200138682A (ko) 2020-12-10

Similar Documents

Publication Publication Date Title
WO2010044577A2 (fr) Procédé pour la fabrication d’un support poreux tridimensionnel utilisant une poudre dérivée de tissu animal, et support poreux tridimensionnel fabriqué par ce procédé
EP2254608B1 (fr) Compositions d'extrait compartimental pour génie tissulaire
US11135163B2 (en) Peptide hydrogel properties and its applications
Bari et al. Association of silk sericin and platelet lysate: Premises for the formulation of wound healing active medications
US20040191226A1 (en) Method for repair of body wall
JP2002518319A (ja) 粒状無細胞組織マトリックス
US20190134099A1 (en) Human tissue derived compositions and uses thereof
US20040187877A1 (en) Method for repair of liver tissue
US20120115222A1 (en) Composition and method for maintenance, differentiation, and proliferation of stem cells
US20120027732A1 (en) Thermoreversible collagen
EA037396B1 (ru) Модифицированный коллаген
CN109157676A (zh) 一种重塑复合生物羊膜的制备方法
CN115957179A (zh) 一种负载淫羊藿苷的3d培养间充质干细胞外泌体水凝胶的制备方法和应用
WO2020246781A1 (fr) Procédé de micronisation de tissu mou vivant, et greffe particulaire de tissu mou vivant ainsi produite
WO2017074093A1 (fr) Matériau de pansement comprenant une matrice dermique acellulaire fibrillée et un polymère biodégradable et son procédé de préparation
US20010006813A1 (en) Methods and compositions for the preparation of cell transplants
US20220218868A1 (en) Novel polysaccharide-based hydrogel scaffolds for wound care
WO2021125373A1 (fr) Composition médicale comprenant une matrice extracellulaire issue du tissu adipeux, et procédé de préparation associé
KR102123903B1 (ko) 분화인자가 함유된 엑소좀 나노 캐리어 조성물 및 이의 제조방법
WO2021177503A1 (fr) Composition utilisant une encre biologique à base de composant cartilagineux pour construire une structure à des fins de traitement de microtie, et procédé de préparation associé
ITVR990082A1 (it) Substrato bioartificiale per la realizzazione di tessuti e organianimali, in particolare umani.
RU2452527C1 (ru) Способ регенерации хрящевой гиалиновой ткани суставов на начальных стадиях деструктивных заболеваний
WO2023085453A1 (fr) Procédé de production d'une matrice dermique acellulaire ultrafine dérivée de la peau animale
WO2013002553A2 (fr) Procédé de prolifération en vue d'une utilisation dans une transplantation de cartilage de ménisque
WO2021117915A1 (fr) Échafaudage utilisant une matrice extracellulaire dérivée de tissus adipeux, et son procédé de production

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20818600

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 29.04.2022)

122 Ep: pct application non-entry in european phase

Ref document number: 20818600

Country of ref document: EP

Kind code of ref document: A1