WO2017195225A1 - Procédé d'extraction et de séparation de cellules souches dérivées de tissus adipeux pour des traitements esthétiques - Google Patents

Procédé d'extraction et de séparation de cellules souches dérivées de tissus adipeux pour des traitements esthétiques Download PDF

Info

Publication number
WO2017195225A1
WO2017195225A1 PCT/IT2016/000122 IT2016000122W WO2017195225A1 WO 2017195225 A1 WO2017195225 A1 WO 2017195225A1 IT 2016000122 W IT2016000122 W IT 2016000122W WO 2017195225 A1 WO2017195225 A1 WO 2017195225A1
Authority
WO
WIPO (PCT)
Prior art keywords
stem cells
composition
fat
barrel
cells
Prior art date
Application number
PCT/IT2016/000122
Other languages
English (en)
Inventor
Egidio Tranfaglia
Valerio MATANÒ
Original Assignee
Promoitalia Group Spa
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 Promoitalia Group Spa filed Critical Promoitalia Group Spa
Priority to PCT/IT2016/000122 priority Critical patent/WO2017195225A1/fr
Publication of WO2017195225A1 publication Critical patent/WO2017195225A1/fr

Links

Classifications

    • 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
    • A61K35/35—Fat tissue; Adipocytes; Stromal cells; Connective tissues
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/64—Containers with integrated suction means
    • A61M1/67—Containers incorporating a piston-type member to create suction, e.g. syringes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M2202/00—Special media to be introduced, removed or treated
    • A61M2202/08—Lipoids

Definitions

  • the present invention relates to methods for aesthetic treatments, in particular it relates to lipofilling technique associated to autologous fat transfer in applications including plastic and reconstructive surgery, neurosurgery, gastrointestinal and affiliated organ surgery, urological surgery, general surgery, orthopedic surgery, gynecological surgery, thoracic surgery, and laparoscopic surgery.
  • plastic and reconstructive surgery including plastic and reconstructive surgery, neurosurgery, gastrointestinal and affiliated organ surgery, urological surgery, general surgery, orthopedic surgery, gynecological surgery, thoracic surgery, and laparoscopic surgery.
  • Autologous fat transfer for aesthetic purposes has evolved to one of the safest and most valuable treatment types available for numerous indications.
  • Autologous fat transfer also known as fat grafting, fat transplantation and lipofilling
  • Numerous lipofilling treatments are often required by women to increase volume and visibly improve the appearance of buttocks, calves, breasts and face. Lipofilling is intended to correct a body shape obviously altered by age, stress, failed diets, excessive weight loss or continuous exposure to solar or artificial UV radiation.
  • breast procedures remain the most popular.
  • MSCs Mesenchymal stem cells
  • ADSCs adipose tissue
  • BMSCs bone marrow derived MSCs
  • Adipose derived stem cell are particularly useful, as they can differentiate into mature adipocytes and into host tissue cells after reimplantation. Hence, the fat graft combined with ASCs is very effective in maintaining the volume of the injected fat tissue.
  • Adipose stem cells have the advantages of superior self-renewal capacity and easy culture ex vivo when compared to marrow-derived mesenchymal stem cells.
  • Advantageously adipose stem cells can be isolated from abundant adipose tissue and can be harvested in a simple and safe manner. From a functional standpoint, adipose stem cells and marrow-derived mesenchymal stem cells were found to share almost the same characteristics, including multipotency, cytological characteristics, immunological characteristics and tissue regeneration.
  • adipose stem cells have been considered as possible replacements for mesenchymal stem cells and have proven their great usefulness in medical applications, moreover, due to such advantages adipose stem cells in some applications are preferred to marrow-derived mesenchymal stem cells in terms of tissue accessibility, stability, effectiveness and economic efficiency.
  • the fat While performing autologous fat transfer, the fat is aspirated from the subcutaneous tissue layer, usually the abdominal wall by means of a suction syringe, and injected into the subcutaneous tissues overlying a depression.
  • Common harvesting techniques include syringe aspiration and vacuum pump aspiration. In practicing such procedures, only part of the injected fat survives and consequently the amount of fat injected strategically has to be in excess respect to that needed for filling the depression.
  • the methods usually include removing adipose tissue from a patient, processing a portion of the adipose tissue to obtain a substantially isolated population of regenerative cells; mixing the regenerative cells with another portion of adipose tissue to form a composition; and administering the composition to the patient from whom the adipose tissue was removed.
  • fat cells purification can be performed by different techniques:
  • centrifugation which can be achieved by lodging directly the syringe into the centrifuge (in this case it is necessary to remove the plunger and cap the two ends of the syringe) , or pouring thelipoaspirate into another container suitable for centrifugation .
  • This is the most reliable technique especially for the low risk of contamination and the possibility of eliminating any residual anesthetic solution which, if re-injected with the cells, may cause side effects such as burning sensation and edema.
  • Centrifugation enables the fractionation of the various components of the aspirated fat according to their specific weight.
  • the different components appear fully distinct and different in colors from the bottom to the top of the container (syringe or test tube) according to the following order from - the bottom upwards :
  • the bottom layer is consisting of a mixture of stromal tissue and ASCs cells
  • the second layer is consisting of physiological and anesthetic solution mixed with blood and vascular residues ;
  • the third layer is consisting of adipose tissue; and the top layer is consisting of oily substance essentially spilled material from traumatized fat cells.
  • anesthetic solution and stromal cells have to be removed. After centrifugation stromal cells, due to their high specific weight, remain adherent to the bottom of the syringe/tube forming a .compact lenticular deposit. During the first phase of expulsion, because the force exerted by the syringe piston, the stromal component crumbles mixing up the anesthetic component. Then the intact fat cells are harvested and finally the oily component containing the fat cells debris are eliminated .
  • adipocytes In order to increase the amount of adipocytes available to be re-implanted many methods have been proposed to isolate and culture adipocytes. According to a typical method, after fat is sucked out or excised and crushed to pieces, its tissue is digested with collagenase, followed by centrifugation . Then, the sediment is collected and a stromal vascular fraction (SVF) is separated therefrom. The SVF is used for cell culture.
  • SVF stromal vascular fraction
  • PCT International Publication No. WO2005/042730 suggests a method for preparing stem cells without collagenase treatment, the method comprising: A) obtaining an aspirate by liposuction; B) transferring the aspirate to a centrifuge to obtain a cell fraction; C) subjecting the cell fraction to centrifugation by specific gravity; and D) collecting a cell layer with lower specific gravity than that of erythrocytes .
  • the European patent application EP 2503007 discloses a method for isolating stem cells by using a specially designed apparatus including: a container containing an aspirate; a piston having an outer diameter corresponding to the inner diameter of the container and having at least one through-hole; and a connection tube adapted to feed an enzyme or a washing solution into the container through the through-hole, having a tip connected to the through-hole, and connected to an external tube or another container containing the enzyme or washing solution at the other end thereof.
  • the method includes pulling the piston backward to form a negative pressure in the container containing the aspirate and to allow the enzyme or washing solution to enter the container containing the aspirate through the connection tube and the through- hole of the piston.
  • LLLI Low-level laser irradiation
  • the adipose isolated cells have to be cultured prior to be irradiated, forcing the isolated cells to a further step wherein they can come in contact with air, ingredients and/or reagent and possible contaminants, hence such passage can consistently affects the final success rate.
  • the international application WO 2009/073724 discloses a method and an apparatus for isolating cells from lipoaspirate that includes a lipid separating apparatus having one or more dispersing parts equipped with a plurality of pores, or other means of dispersion, and a cell separation assembly including a plurality of optionally removable filters of variable pore size.
  • the relative method enables to prepare a population of cells for cell transplantation into a patient in the need thereof including dissociating a sample of donor adipose tissue into individual cells and small clusters of cells until the dissociated cells and clusters are reduced in diameter, separating the individual cells and small clusters of cells into an aqueous cellular layer and a lipid layer without centrifugation, collecting cells from aqueous cellular layer for cell transplantation including stromal vascular cells.
  • the Lipogems ⁇ device includes at least one washing and separating container having a washing chamber; the container has an inlet through which the liposuctioned material enters the washing chamber, and an outlet through which at least part of the material leaves the washing chamber, the washing chamber includes stirring means for forming an emulsion of fluid components.
  • the adipose tissue obtained by this method includes a fluid component that have an oily component, a blood component and/or sterile solutions, and a solid component including cell fragments, cells and one or more cell macroagglomerates of heterogeneous size.
  • the stirring or agitation means are balls that moving within the digestion chamber agitate the fat agglomerates and facilitate the release of the cells.
  • the device provides tissue for transplantation without using chemicals for preparation, i.e. with no chemical aggression or any other chemical treatment of the lipoaspirate, however the system cannot prevent cell injury due to mechanical action they are subjected to, and the extensive washings necessary to remove the excess fluid (i.e. anesthetic and blood cells).
  • the international patent application WO2013/025869 relates to the separation by centrifugation of adipose tissue from aspirated tissues wherein firstly a tumescent fluid is injected into the area from which the adipose tissue has to be removed, then adipose tissue is aspirated by a needle or cannula inserted into the area where the tumescent fluid has been injected and applying a vacuum.
  • the fat and tumescent fluids are then typically allowed to partially fractionate by standing in a tube or syringe, shown in FIG.l, before (A) and after (B) centrifugation, whereby the fractions of different densities separate by gravity.
  • the syringe 2 that is also initially used to aspirate the fluids from the patient, is placed in a centrifuge to separate the adipose tissue from the tumescent fluid according to their densities.
  • the syringe 2 is provided with an element 22 designed to float above the layer of adipose tissue 20 after centrifugal separation.
  • the floating element 22 is made of a material that will absorb the oils that separate from the aspirated fluid 6 during centrifugation and at least partially retain it in the element to prevent remixing with the adipose tissue during handling of the container and removal of the separated fluids from the container.
  • the centrifugal forces separate the tumescent fluid 16 from the adipose tissue 18, and oil from damaged adipose cells separates as a third, least and dense fraction above the adipose tissue layer 20.
  • the relative method comprises the step of aspirate adipose into a syringe having a cavity acting as separating chamber wherein the aspirated adipose fluids are separated into components based on relative densities and having therein a disk movable within said cavity made of materials and configured such that it floats on a layer of adipose tissue that has been separated from oils released from damaged adipose cells and prevents substantially remixing of said oils with said adipose during handling of the container/syringe.
  • the oil-absorbing element made of a preferably porous material, and having a density such that it automatically positions itself between the adipose-cells fraction and the less dense oil fraction after centrifugation and such that during centrifugation, as the less dense fraction of oil is forming, the oil is entrained in the floating element, the undesired remixing between the oil back and the adipose tissues cannot be excluded. Furthermore, it cannot be excluded also that the sedimentation of adipose cells or small aggregates of adipose cells in the bottom layer containing excess fluid will be lost with the expulsion of this first fraction .
  • ASC mesenchymal stem cells
  • the enzyme treatment if provided, can cause damage to cells and affect the subsequent cell re- implant, especially if not completely removed;
  • a first object of the present invention is to provide a method for processing ex vivo the lipoaspirate tissue from a subject and transferring the obtained adipose tissue for aesthetic purposes in an autologous way. Said method allows to obtain a homogeneous fraction of adipose tissue enriched with adipose derived stem cells, while minimizing damage to adipose tissue.
  • Another object of the present invention is to provide a kit for isolating stem cells and fat tissue from a lipoaspirate and producing a homogeneous fraction of adipose tissue enriched with adipose derived stem cells to be reimplatend according to the method of the invention.
  • composition promoting pre-adypocites proliferation and differentiation into mature adypocites.
  • Figure 1 is a cross section view of a prior art container/syringe with aspirated adipose fluid before density fractionation (A) and after density fractionation (B) .
  • Figure 2 is a side view of the barrel in accordance with the invention with aspirated adipose fluid stratified after density fractionation.
  • Figure 3 is a side view of the barrel in accordance with the invention during extraction by syringe of the several components emerged and separated after density fractionation.
  • Figure 4 is an operational flow chart of the method for preparing a homogeneous fraction of adipose tissue enriched with adipose derived stem cells for lipofilling techniques according to the invention.
  • Figure 5 is an alternative operational flow chart of the method for preparing a homogeneous fraction of adipose tissue enriched with adipose derived stem cells for lipofilling techniques according to the invention.
  • Figure 6 shows immunohistochemical analysis performed on cells isolated according to the method of the invention.
  • Figure 7 shows differentiated adypocites obtained according to the method of the invention.
  • the principle of operation basing the method according to the invention is the collection of adipose tissue from one location of the patient body by a syringe, transfer of the tissue into the a container configured to subjected to centrifugation .
  • the centrifugation process allows the separation of the various cellular components of the collected adipose tissue.
  • a mixture of adipose derived stem cells (ACSs) and adipose tissue to be transplanted into patients in autologous way for aesthetic treatments is provided.
  • the method uses fat tissue as starting material, which can be obtained by liposuction through aspiration performed by using a syringe attached to an aspirating needle of conventional design, which are not part of the invention.
  • the latch lock integrated syringe 40 (not shown in detail) lock withdrawn plunger into fixed relation with barrel to physically block the plunger in place maintaining the negative suction pressure, as known from the prior art, so that the suction pressure can be retained without the need to manually hold the plunger in a desired withdrawn position.
  • the syringe barrel presents an inward protruding stop pin wherein the plunger fins engage; so, after fat tissue aspiration, in order to create and maintain vacuum the syringe plunger has to be withdrawn to the desired position and to set the syringe to lock, at the desired position the plunger has to be rotated clockwise so that the locking fins can be engaged behind the stop pin. To release the syringe plunger has to be counterclockwise turned.
  • the needle is removed and the syringe barrel by means of a luer-lock connector 43 is connected to a tube 3, or cylindrical body container, or barrel, wherein the lipoaspirate is transferred (FIG. 2) .
  • Said barrel 3 acts as a separation chamber wherein the adipose aspirate fluids separate into components thereof based on relative densities by centrif gation .
  • lipoaspirate as herein used, are intended to include all substances obtained by liposuction, and includes adipose tissue and biological material sucked out by liposuction .
  • the barrel 3 has capped openings 31, 32 at each end, reversibly closable by pressure and/or screw caps.
  • the bottom cap 32 is a pressure cap
  • the top cap 31 is a screw cap.
  • the barrel 3 is configured to be subjected to centrifugation .
  • the aspirate in the tube is fractionated by centrifugation performed at 1500-4500 rpms for 5-15 minutes, preferably centrifugation is performed at 3000 rpms for 8 minutes.
  • the aspirated material contained iri the tube is fractionated showing different layers containing the various components of the adipose tissue readily identifiable.
  • the layers appear in the tube from bottom to top in the following order: mesenchymal stem cells and stromal tissue 23, saline mixed to vascular residues and anesthetic 16, fat cells 18 and, on the top of the barrel is layered the oil component 20 from damaged adipose tissue (FIG. 2) .
  • the top cap 31 is removed and a 3 ml syringe 40 is connected through a luer lock connector 43 to the top opening of the barrel 3; then the bottom pressure cap 32 is removed and connected to a cylindrical screw plunger 41.
  • Said screw plunger 41 has the outer diameter corresponding to the inner diameter of the barrel, through a precise and slow forwardly screwing movement, it causes the outflow of the oily component stratified in the upper part of the tube 3 from the top opening thereof.
  • the fractions stratified in the barrel are extracted by several syringes 40, in turns attached by a luer lock connector 43, to the upper opening of the cylindrical tube in the following order: firstly, the oily component is removed and discharged, as above described. Then, another syringe 40 is connected by the luer-lock 43 to the barrel 3 to collect the adipose cells. Then, as the screw plunger has gradually advanced, by a further syringe 40 connected to the barrel 3 the physiological solution and vascular residues are removed and discarded.
  • adipose tissue fraction previously collected is then reintroduced to the barrel 3 to be mixed with the elements of the last layer 23 in the barrel 3, containing stem cells and stromal tissue (Fig. 2) .
  • Such mixture of adipose tissue with stem cells and stromal tissue is then transferred to a further syringe, ready for autologous implantation into the patient .
  • the remarkable advantage provided by the use of the method of the invention is enabling an improved recovery rate of intact and undamaged adipose derived stem cells following the centrifugation process.
  • This technical feature is accomplished thanks to the introduction in the method of the separation step by centrifugation performed, in the specifically configured barrel acting as separation chamber.
  • the specific position of the openings of the barrel wherein the separation is performed and the specific orientation of barrel during the centrifugation contribute to obtain the beneficial results of the method.
  • the method according to the invention differs from other prior art methods known and applied for harvesting and concentrating autologous adipose tissue using a container for centrifuging aspirated adipose fluid, preferably a syringe including a cap sealing the end of the syringe against leakage during centrifugation as in the device shown in Fig. 1.
  • a container for centrifuging aspirated adipose fluid preferably a syringe including a cap sealing the end of the syringe against leakage during centrifugation as in the device shown in Fig. 1.
  • a container for centrifuging aspirated adipose fluid preferably a syringe including a cap sealing the end of the syringe against leakage during centrifugation as in the device shown in Fig. 1.
  • the fluid port end 14 which the several phases emerged are removed from is positioned at the bottom of the container 2.
  • the so obtained stem cells along with the stromal component undergo reshuffling with previously extracted adipose tissue.
  • a significant amount of stem cells alive therefore with a consistent functionality and capacity of engraftment, is mixed with pure fat that has retained its structural architecture.
  • the mixture of stem cells and fat tissue obtained according to the present method comprises stem cells in amount 10% to 90% of the total volume, and fat tissue in amount 10% to 90% of the total volume.
  • the relative percentage of the two components, i.e. stem cells and fat tissue, of the mixture to be infiltrated depends on the feature of the receiving site and of the patient who has to receive the autologous implant.
  • the mesenchymal stem cells isolated by the method of the invention are differentiated stem cells which can develop into adipocyte cells, or in the cells of the host tissue, as a result of autocrine and paracrine type hormonal stimulation; therefore the introduction of a conspicuous amount of this cell type in the autograf is very important to ensure the regeneration of the adipose tissue and a longer sub- dermically permanence of the tissue.
  • the cell mixture so obtained generates in situ an optimal three-dimensional cellular structure able to promote revascularization of the implant in the receiving site and greater longevity thereof.
  • the method according to the invention can optionally involve the addition of a composition acting as enhancer to the mixture of stem cells and fat tissue in order to promote preadipocyte differentiation, and maturation, into adipocyte.
  • a composition acting as enhancer to the mixture of stem cells and fat tissue in order to promote preadipocyte differentiation, and maturation, into adipocyte.
  • Another object of the present invention is to provide a composition promoting adipocytes proliferation and differentiation which according to the method of invention can optionally be added and mixed to the mixture of fat and stem cells.
  • the composition promoting preadipocytes proliferation and differentiation according to the invention comprises among other ingredients: Insulin like growth factor 1, SH-oligopeptide-2 , calcium chloride. - " ⁇ - ⁇ '
  • EXAMPLE 1 Composition promoting preadipocytes proliferation and differentiation
  • composition has the following formulation, wherein the amount of each component is reported as percentage w/w:
  • the ratio of the composition added to the mixture of stem cells and fat tissue is ranging between 0.1:5.0, preferably the ratio is 0.5:3.0, as 0.5 ml of composition are added to 3 ml of stem cells enriched fat tissue.
  • the mixture of stem cells, fat tissue and the composition promoting adipogenic conditions, i.e. promoting pre-adypocites proliferation and differentiation in the receiving site, are gently reshuffled by activation of the syringe plunger before to be implanted in the final receiving site.
  • the introduction of a step activating, and/or increasing, their proliferation in the lipofilling procedure could be desirable; however the activation means known in the art can often damage the cells, hence, reducing the amount of implantable calls and affecting the final outcome of the entire procedure, such as for example by using low level laser irradiation, especially if the laser parameters are not properly controlled.
  • the stimulation of the mesenchymal cells to be implanted in the lipofilling procedure using the provided composition has demonstrated to allow the best performance and outcome of the application of the present method in the regenerative therapy, tissue engineering and aesthetic medicine .
  • the method comprises and optional step wherein the mixture of stem cells and fat tissue prior to be implanted is mixed with the adipogenic composition comprising according to the invention Insulin like growth factor 1, SH-oligopeptide-2 and calcium chloride
  • the present invention provides a method for preparing autologous adipose tissue for use in aesthetic lipograft procedure allowing isolating mesenchymal stem cells from a sample of lipoaspirate, sepatating away unwanted inflammatory oil, lipids and excess fluid.
  • the method further allows to increase the differentiation rate of the isolated stem cells into mature adipocytes, so as the resulting purified adipose concentrate can enable optimal structural grafting and implanting of the mixture of stem cells and fat tissue obtained by the invention into a receiving site of the patient comprising the following steps :
  • composition comprising the association of Insulin like growth factor 1, SH- oligopeptide-2 , calcium chloride to the mixture of the fat and stem cells;
  • kit for the preparation of autologous adipose tissue for use in aesthetic lipograf procedures comprising:
  • the method of the invention before adding the lyophilized composition promoting adipocytes proliferation and differentiation in the mixture of stem cells and fat tissue it is reconstituted with the provided sterile solution and then is added in the ratio of 0.5 ml of composition to 3 ml of stem cells enriched fat tissue.
  • the cell samples obtained by the lipoaspirate according to the method of the invention were washed with saline phosphate buffered solution (PBS) containing 5% penicillin/streptomycin (P/S) to remove debris and anesthetic residues.
  • PBS saline phosphate buffered solution
  • P/S penicillin/streptomycin
  • Cells were subsequently treated with 0.075% collagenase type I and then maintained in culture in DMEM medium, 10% FBS, 1% P/S, at 37 °C, 5% C0 2 . (Pittenger, 1999; Chamberlain, 2007) and two cell cultures were set up.
  • mesenchymal stem cells grow adhering on culture plates and express the surface markers: CD105, CD73 and CD90 in rate higher than 95% and beta- integrins.
  • the identification of surface markers is performed by immunohistochemistry techniques targeting the specific antigens for adult mesenchymal stem cells.
  • beta-integrins were used as identification markers for adult mesenchymal stem cells .
  • the cells from the lower layer stratified in the barrel following centrifugation, were deposited on the microscope slide through a cytocentrifugation and fixed in 10% buffered formalin solution.
  • a specific antibody for beta-integrin 1 was used at a dilution of 1:100 for 1 hour at room temperature.
  • the Avidin-Biotin Complex (ABC) staining was used using diaminobenzidine as colorant and hematoxylin as the nuclear contrasting.
  • the results are illustred in Figure 6 showing four independent examples of positivity for the expression of beta integrin 1, considered as a typical marker of adipose stem cells, in cells isolated according to the method. 2.
  • stem cells harvested from a lipoaspirate obtained according to the method of the invention were cultured in the presence and in the absence (control) of the adipogenic condition by addition of the adipogenic composition.
  • Cells were seeded and cultured onto microscope slides. At different time points cells were stained with Oil Red 0 staining. Results are shown in FIG. 7 at lipoaspiration (time 0) (panels A), 6 days after lipoaspiration (panels B) , and 14 days after lipoaspiration (panels C) .
  • Oil Red 0 staining clearly highlighted accumulated lipid droplets in the cells indicating an increased differentiation rate under adipogenic conditions.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biomedical Technology (AREA)
  • Cell Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Immunology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Developmental Biology & Embryology (AREA)
  • Chemical & Material Sciences (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Biotechnology (AREA)
  • Virology (AREA)
  • Zoology (AREA)
  • Hematology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Vascular Medicine (AREA)
  • Anesthesiology (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé de préparation d'un mélange de cellules souches dérivées de tissus adipeux et de tissus graisseux pour une intervention de liporemplissage dans des traitements esthétiques et un kit associé.
PCT/IT2016/000122 2016-05-10 2016-05-10 Procédé d'extraction et de séparation de cellules souches dérivées de tissus adipeux pour des traitements esthétiques WO2017195225A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/IT2016/000122 WO2017195225A1 (fr) 2016-05-10 2016-05-10 Procédé d'extraction et de séparation de cellules souches dérivées de tissus adipeux pour des traitements esthétiques

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/IT2016/000122 WO2017195225A1 (fr) 2016-05-10 2016-05-10 Procédé d'extraction et de séparation de cellules souches dérivées de tissus adipeux pour des traitements esthétiques

Publications (1)

Publication Number Publication Date
WO2017195225A1 true WO2017195225A1 (fr) 2017-11-16

Family

ID=56682218

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IT2016/000122 WO2017195225A1 (fr) 2016-05-10 2016-05-10 Procédé d'extraction et de séparation de cellules souches dérivées de tissus adipeux pour des traitements esthétiques

Country Status (1)

Country Link
WO (1) WO2017195225A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110038165A (zh) * 2019-04-24 2019-07-23 成都远山原力生物科技有限公司 一种富集高活性脂肪颗粒细胞和脂肪干细胞的脂肪移植物及其制备方法和应用
CN113041397A (zh) * 2021-04-08 2021-06-29 红色未来科技(北京)有限公司 一种含有交联葡聚糖的面部填充剂及其制备方法
IT202000002599A1 (it) * 2020-02-10 2021-08-10 Promoitalia Group Spa “siringa dotata di mezzi per la separazione selettiva dei componenti di una miscela eterogenea multifase, in particolare delle cellule staminali derivate da tessuto adiposo, kit relativo, e metodo per il suo impiego”
CN114653448A (zh) * 2022-03-25 2022-06-24 南京弗瑞莱贸易有限公司 一种脂肪微粒化装置及使用方法

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005042730A2 (fr) 2003-11-04 2005-05-12 Biomaster, Inc. Procede et systeme pour preparer des cellules souches a partir de tissus gras
WO2009073724A1 (fr) 2007-12-04 2009-06-11 Ingeneron, Inc. Appareil et procédé pour l'isolation de cellules
EP2503007A1 (fr) 2009-11-17 2012-09-26 Hee-Young Lee Procédé pour la séparation de cellules souches
US20130034524A1 (en) * 2011-08-03 2013-02-07 Siamak Agha-Mohammadi Non-Enzymatic Method for Harvesting Adipose-Derived Stromal Cells and Adipose-Derived Stem Cells from Fat and Lipo-Aspirate
WO2013025869A1 (fr) 2011-08-17 2013-02-21 Harvest Technologies Corporation Ségrégation d'huiles dans le fractionnement de tissus adipeux aspirés
US20130087643A1 (en) * 2010-03-23 2013-04-11 Carlo Tremolada Device and a method for preparing a tissue
WO2014207135A1 (fr) * 2013-06-26 2014-12-31 Stemform Aps Opération de lipofilling à l'aide de cellules souches dérivées du tissu adipeux expansées ex vivo pour augmentation mammaire ou pour comblement et/ou rajeunissement facial à des fins esthétiques
US20150231641A1 (en) 2010-05-20 2015-08-20 Lipogems International S.P.A. Device and method for preparing tissue, particularly adipose tissue
WO2016067246A1 (fr) * 2014-10-31 2016-05-06 Eltek S.P.A. Dispositif médical pour la préparation d'un concentré de cellules

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005042730A2 (fr) 2003-11-04 2005-05-12 Biomaster, Inc. Procede et systeme pour preparer des cellules souches a partir de tissus gras
WO2009073724A1 (fr) 2007-12-04 2009-06-11 Ingeneron, Inc. Appareil et procédé pour l'isolation de cellules
EP2503007A1 (fr) 2009-11-17 2012-09-26 Hee-Young Lee Procédé pour la séparation de cellules souches
US20130087643A1 (en) * 2010-03-23 2013-04-11 Carlo Tremolada Device and a method for preparing a tissue
US20150231641A1 (en) 2010-05-20 2015-08-20 Lipogems International S.P.A. Device and method for preparing tissue, particularly adipose tissue
US20130034524A1 (en) * 2011-08-03 2013-02-07 Siamak Agha-Mohammadi Non-Enzymatic Method for Harvesting Adipose-Derived Stromal Cells and Adipose-Derived Stem Cells from Fat and Lipo-Aspirate
WO2013025869A1 (fr) 2011-08-17 2013-02-21 Harvest Technologies Corporation Ségrégation d'huiles dans le fractionnement de tissus adipeux aspirés
WO2014207135A1 (fr) * 2013-06-26 2014-12-31 Stemform Aps Opération de lipofilling à l'aide de cellules souches dérivées du tissu adipeux expansées ex vivo pour augmentation mammaire ou pour comblement et/ou rajeunissement facial à des fins esthétiques
WO2016067246A1 (fr) * 2014-10-31 2016-05-06 Eltek S.P.A. Dispositif médical pour la préparation d'un concentré de cellules

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BRIAN M STREM ET AL: "Multipotential differentiation of adipose tissue-derived stem cells", KEIO JOURNAL OF MEDICINE., vol. 54, no. 3, 1 January 2005 (2005-01-01), JP, pages 132 - 141, XP055339415, ISSN: 0022-9717, DOI: 10.2302/kjm.54.132 *
KURITA MASAKAZU ET AL: "Influences of centrifugation on cells and tissues in liposuction aspirates: optimized centrifugation for lipotransfer and cell isolation", PLASTIC AND RECONSTRUCTIVE SURGERY, WOLTERS KLUWER HEALTH, US, vol. 121, no. 3, 1 March 2008 (2008-03-01), pages 1033 - 1041, XP008119618, ISSN: 0032-1052, DOI: 10.1097/01.PRS.0000299384.53131.87 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110038165A (zh) * 2019-04-24 2019-07-23 成都远山原力生物科技有限公司 一种富集高活性脂肪颗粒细胞和脂肪干细胞的脂肪移植物及其制备方法和应用
CN110038165B (zh) * 2019-04-24 2021-09-28 成都远山原力生物科技有限公司 一种富集高活性脂肪颗粒细胞和脂肪干细胞的脂肪移植物及其制备方法和应用
IT202000002599A1 (it) * 2020-02-10 2021-08-10 Promoitalia Group Spa “siringa dotata di mezzi per la separazione selettiva dei componenti di una miscela eterogenea multifase, in particolare delle cellule staminali derivate da tessuto adiposo, kit relativo, e metodo per il suo impiego”
CN113041397A (zh) * 2021-04-08 2021-06-29 红色未来科技(北京)有限公司 一种含有交联葡聚糖的面部填充剂及其制备方法
CN114653448A (zh) * 2022-03-25 2022-06-24 南京弗瑞莱贸易有限公司 一种脂肪微粒化装置及使用方法
CN114653448B (zh) * 2022-03-25 2024-03-12 湖南锦泓医疗科技有限公司 一种脂肪微粒化装置及使用方法

Similar Documents

Publication Publication Date Title
US11389616B2 (en) Method for preparing tissue, particularly adipose tissue, for transplantation from lobular fat extracted by liposuction
US10617812B2 (en) System and method for obtaining a cellular sample enriched with defined cells such as platelet rich plasma (PRP)
KR102369602B1 (ko) 지방 조직 원심 분리기 및 그 사용 방법
US10537596B2 (en) Bone marrow adipose portion isolation device and methods
BR112013015215B1 (pt) Método para recuperação facilitada de células regenerativas a partir de uma amostra de tecido do corpo
WO2017195225A1 (fr) Procédé d'extraction et de séparation de cellules souches dérivées de tissus adipeux pour des traitements esthétiques
US8673639B2 (en) Methods for isolating stem cells
US20240010967A1 (en) Apparatus and method for isolating stem cells
KR101719743B1 (ko) 지방 조직으로부터 지방줄기세포를 수득하는 방법
US20160272943A1 (en) Apparatus and method for closed system recovery of cells from tissue samples
TW202026416A (zh) 分離血液的方法
KR101013927B1 (ko) 줄기세포 분리방법
CN111849886B (zh) Svf细胞以及制备方法与应用
Abul Hassan et al. Facial Cell Assisted Lipograft Comparing Mechanical and Enzymatic Preparation of the Stromal Vascular Fraction: A Comparative Study

Legal Events

Date Code Title Description
NENP Non-entry into the national phase

Ref country code: DE

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

Ref document number: 16750497

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 16750497

Country of ref document: EP

Kind code of ref document: A1