MATANÒ VALERIO (IT)
| WO2013025869A1 | 2013-02-21 | |||
| WO2016067246A1 | 2016-05-06 | |||
| WO2014207135A1 | 2014-12-31 | |||
| WO2005042730A2 | 2005-05-12 | |||
| WO2009073724A1 | 2009-06-11 | |||
| WO2013025869A1 | 2013-02-21 |
| US20130034524A1 | 2013-02-07 | |||
| US20130087643A1 | 2013-04-11 | |||
| EP2503007A1 | 2012-09-26 | |||
| EP2503007A1 | 2012-09-26 | |||
| US20150231641A1 | 2015-08-20 |
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
| CLAIMS 1) A method for preparing ex vivo a mixture of adipose derived stem cells and fat tissue for lipofilling procedure comprising: a) obtaining a lipoaspirate from liposuction performed by controlled negative pressure syringe (40) ; b) transferring the lipoaspirate into a barrel (3) configured to be subjected to centrifugation and having at the top end a reversible screw capped opening (31) and at the bottom a reversible pressure capped opening (32) ; c) separation of the lipoaspirate fractions by centrifugation at 1500-4500 rpms for 5-15 minutes ; d) assembling the barrel (3) with a series of syringes (40) connected one by one through a luer lock connector (43) to the top opening (31) of the barrel (3) and a cylindrical screw plunger (41) having the outer diameter corresponding to the inner diameter of the barrel (3) at the bottom opening (32) of the barrel; e) collecting of the lipospirate fractions; f ) mixing stem cells and fat tissue obtained from the relative fractions (20, 18, 16, 23); 2) Method according to claim 1 wherein the centrifugation is performed at 2500-3000 rpms for 8 minutes . 3) Method according to claims 1 and 2 wherein in the mixture of stem cells/fat tissue stem cells are in amount 10% to 90% of the total volume, and fat tissue are in amount 10% to 90% of the total volume. 4) Method according to any of the preceding claims further comprising: g) addition of a composition comprising the association of Insulin like growth factor 1, SH-oligopeptide-2 , calcium chloride to the mixture of the fat and stem cells obtained from step f ) . 5) Method according to claim 4 wherein the composition comprises: SH-oligopeptide-2 0.1-2 % w/w Insulin like growth factor 1 0.1-2 % w/w Calcium chloride 0.1-1 % w/w Calcium gluconate 0.1-6 % w/w Sodium hyaluronate 0.1-1 % w/w Glucose 0.1-2 % w/w Glycerin 0.01-0.1 % w/w Phenoxyethanol 0.01-0.1 % w/w Disodium EDTA 0.01-0.1 % w/w Glycine soja oil 0.01-0.1 % w/w Sodium oleate 0.01-0.1 % w/w Hydrogenated lecithin 0.01-0.1 % w/w Deionized water Up to 100 g. 6) Method according to claims 4 and 5 wherein the composition has the following formulation: SH-oligopeptide-2 1,5 % w/w Insulin like growth factor 1 1 % w/w Calcium chloride 0.8 % w/w Calcium gluconate 4 % w/w Sodium hyaluronate 0.8 % w/w Glucose 1.5 % w/w Glycerin 0.05 % w/w Phenoxyethanol 0.05 % w/w Disodium EDTA 0.05 % w/w Glycine soja oil 0.05 % w/w Sodium oleate 0.05 % w/w Hydrogenated lecithin 0.05 % w/w Deionized water Up to 100 g. 7) Method according to any of claims 4-6 wherein the composition is added to the mixture of stem cells and fat tissue in the ratio of composition: mixture equal to 0.5:3.0. 8) Kit for preparing a mixture of adipose derived stem cells and fat tissue for lipofilling procedure comprising: 1 sterile surgical cover cloth 50x50 cm; 3 3 ml syringes with integrated latch lock (luer lock) ; 1 30 ml syringe (luer lock); 1 10 ml syringe (luer lock) ; 2 20, 50, 100 ml barrels; 2 screw plungers; 6 luer-lock connectors; 3 needles or cannulas. 9) Kit according to claim 8 further comprising: 2 vials containing the lyophilized composition promoting adipocytes proliferation and differentiation; 2 vials containing 1ml of sterile saline. 10) Composition comprising: SH-oligopeptide-2 0.1-2 % w/w Insulin like growth factor 1 0.1-2 % w/w Calcium chloride 0. 1-1 % w/w Calcium gluconate 0. 1-6 % w/w Sodium hyaluronate 0. 1-1 % w/w Glucose 0. 1-2 % w/w Glycerin 0. 01- 0.1 % w/w Phenoxyethanol 0. 01- 0.1 % w/w Disodium EDTA 0. 01- 0.1 % w/w Glycine soja oil 0. 01- 0.1 % w/w Sodium oleate 0. 01 - 0.1 % w/w Hydrogenated lecithin 0. 01- 0.1 % w/w Deionized water Up ) to 100 g. 11) Composition according to claim 10 having the following formulation: SH-oligopeptide-2 1 , 5 % w/w Insulin like growth factor 1 1 % w/w Calcium chloride 0.8 % w/w Calcium gluconate 4 % w/w Sodium hyaluronate 0.8 % w/w Glucose 1.5 % w/w Glycerin 0.05 % w/w Phenoxyethanol 0.05 % w/w Disodium EDTA 0.05 % w/w Glycine soja oil 0.05 % w/w Sodium oleate 0.05 % w/w Hydrogenated lecithin 0.05 % w/w Deionized water Up to 100 g. 12 ) Use of the composition of claims 10 and 11 for promoting proliferation and differentiation ex vivo of isolated pre-adypocites in the lipofilling procedure . |
DERIVED FROM ADIPOSE TISSUE FOR EASTHETIC TREATMENTS
~k ~k ~k
Field of the invention
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. Background of the invention
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, is becoming a popular procedure among physicians and patients, it has been proven in both scientific literature and every day practice to provide a wide-range of treatments, from treatment of tissue injuries and defects to adipose tissue based wound sealing, from breast reconstruction and augmentation to facial aesthetics, as well as volumization and as an adjunct for body shaping procedures. 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. However, as the range of indications for fat transfer grows, breast procedures remain the most popular.
Success rates of autologous fat grafting have also improved greatly in recent years, with decreased complications and better quality technology to harvest and re-inject fat. In addition, an admirable progress of investigating new techniques related to the process of harvesting and preparation of fat cells, storage, injection techniques and the biological characteristics of the recipient site has been achieved .
Hence, the advent of liposuction techniques, the abundant donor-tissue availability and the relative ease of harvesting has made autologous fat an attractive material for use as soft-tissue filler.
Furthermore, recent studies have shown that adipose tissue can be considered a valuable source of stem cells. Mesenchymal stem cells (MSCs) derived from adipose tissue (ADSCs) have a differentiation potential similar to that of bone marrow derived MSCs (BMSCs) and can differentiate into different cell types such as adipocytes, chondrocytes, osteoblasts, and myoblasts.
Starting from this assumption, the modern techniques of tissue engineering have opened the way for a more refined and sophisticated use of lipofilling .
Adipose derived stem cell (ACSs) 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. Therefore, 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.
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.
Method for augmenting autologous fat transfer have been recently developed. 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.
Generally, fat cells purification can be performed by different techniques:
- by decanting, which provides the advantage of not traumatize the fat cells, but requires long operative time and, therefore, increasing the risk of oxidation of fat cells exposed to air.
- by filtration, which can be performed both through metal filters (not recommended choice) and through gauze which exposes also to the risk of contamination.
by washing, generally performed by using Ringer's lactate solution. Indeed this method is not commonly exploited as it is believed that it could damage the adipocyte both mechanically and osmotically.
- by 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.
From the above it is evident that it is necessary to isolate an adipocytes fraction as much pure as possible to be transplanted in order to decrease the inflammatory response after re-implanting; if in the recipient site there are many cellular debris an intense inflammatory reaction develops with consequent activation of the inflammatory reaction.
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.
However, the above-mentioned filtration technique, provide greater survival of the stromal cells (ASCs) , nevertheless the procedure is extremely laborious and does not guarantee sterility.
In order to collect the adipose tissue, 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 .
Through the instrumentations commonly used, or the common syringes, it is not possible to obtain a good cell preparation characterized by clear separation of the components in the fat aspirate. Even experienced operators find difficult to measure out exactly the force of the injection to avoid the mixing up of the separated phases.
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.
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.
Furthermore, studies have shown that the higher the proliferation of MSCs, the greater the regenerative and healing capacity of the tissues where they reside. Low-level laser irradiation (LLLI) has been shown to be effective in a variety of medical conditions such as mucosal healing, skin ulcers, dermatitis, and mucositis by exerting positive biomodulatory effects on MSCs. This capacity of accelerating the healing process is most likely related to the finding that LLLI promotes cell proliferation .
However, to increase the proliferation of MSCs or ADSCs, and consequently the regenerative and healing capacity of the recipient tissues where they will reside after re-implantation, 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.
An alternative single-use kit and relative method for the lipoaspiration, processing and deployment of adipose tissue is described in US 2015/0231641 and available under the commercial name Lipogems©. The entire process is carried out in one surgical step. According to this method the procedure is achieved in a sealed, sterile device, allowing a gradual reduction of the adipose clusters and the elimination of oily and hematic pro-inflammatory content. The entire process is performed with immersion in a saline solution minimizing any trauma to the cellular products .
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.
In such a way 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).
Introducing a centrifugation step in the procedure for producing adipocytes to be implanted improves the separation and the cell recovery rate.
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. It has also been found useful in subsequent handling and in order to improve the quality of the adipose tissue processing the aspirated fluid in a centrifuge. Therefore, 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. According to this invention, 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.
However, according to the structure of the syringe acting as separating chamber, critical appear to be the discharging operations of the several fractions from the syringe. First of all tumescent fluids are discharged, then the adipose tissues. Some of such adipose tissues will be damaged in the aspiration and subsequent processing, whereby releasing some oil, which separates as a third layer above the adipose tissues. Despite the presence of 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 .
Therefore, the devices and methods currently available have several drawbacks in that:
- do not allow a proper separation of the fat cells from the oily component and the physiological solution;
do not allow to recover in a rapid and/or absolutely sterile manner mesenchymal stem cells (ASC) ;
- the enzyme treatment, if provided, can cause damage to cells and affect the subsequent cell re- implant, especially if not completely removed;
- proliferation activation, by laser irradiation or growth factors induction, requires a further culture passage potentially affecting the following cell re-implant.
Therefore, it is still strongly felt the need of alternative methods for isolating undifferentiated mesenchymal cells of adipose origin to be employed in the aesthetic treatment involving autologous fat grafting into various parts of the body.
Summary of the invention The present invention has been made in an effort to solve the above problems.
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.
According to another aspect of the invention it provides a composition promoting pre-adypocites proliferation and differentiation into mature adypocites.
Brief description of the drawings
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.
Detailed description of the invention
It is well known that quality of adipose graft material is an important consideration prior to performing any lipograft procedure. While autologous lipografting has become a widely used technique for soft tissue augmentation in aesthetic procedures, indeed, clinical outcomes can be unpredictable. A major challenge is graft volume loss, which can greatly affect aesthetic outcomes. The non proper isolation of the cells to be re-implanted and the presence of excess fluid, oils, and lipids contributes to graft volume loss, as these components are reabsorbed by the body.
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.
According to the method of the present invention 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. According to Fig. 3, 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.
After the syringe 40 has been loaded by aspiration and the plunger has been blocked, while maintaining the negative pressure inside the barrel, 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 .
The expressions "adipose aspirate" and
"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. In particular, the bottom cap 32 is a pressure cap, while the top cap 31 is a screw cap.
According to the invention 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.
After being centrifuged, due to the different relative specific weight of its components, the aspirated material contained iri the tube is fractionated showing different layers containing the various components of the adipose tissue readily identifiable. In particular, 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) .
After centrifugation, 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.
While screwing the plunger to its end run, first of all the oily material is extruded and transferred into the 3 ml syringe 40.
According to the method of this invention by using the above described screw plunger it is possible to prevent the admixture of the layers while extruding, because through screwing steps, in a gradual and precise way, it enables controlling the extrusion force .
Hence, according to the method of the invention 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. The 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. As taught in WO 2013/025869, with reference to the syringe orientation in the operation during and after the centrifugation, the fluid port end 14 which the several phases emerged are removed from, is positioned at the bottom of the container 2. Indeed, during centrifugation a large amount of stem cells are separated from the fat layers because more hydrophilic and heavy, hence the cells remain in the aqueous fraction 16, and are destined to be easily and accidentally lost while removing and discharging the phase containing fluids 16 from the bottom opening the container .
This drawback is overcome by using the method of the invention as there is no loss of adipose derived stem cells forming a sediment at the bottom of the barrel, which are collected by the fat tissue fraction reintroduced in the barrel, ensuring better lipofilling outcomes once re-implanted.
According to the method the so obtained stem cells along with the stromal component undergo reshuffling with previously extracted adipose tissue. In such a way 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.
Hence, 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.
In order to further increase the implant longevity 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. Such partially differentiated adipocytes, derivated by stem cells, duplicate in the receiving site, up to block the mitosis when all the available space has been filled in .
Therefore, 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
The 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.
Because the proliferation of MSCs is usually slow and the yield of these cells after harvesting is also low, 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 .
In fact, a typical problem related to the lipofilling techniques is the reduced permanence of the fat in the replanting site, therefore the enrichment of the fat to be transplanted with stem cells capable of differentiating into new adipocytes, ensures an increased engraftment ability thereof. Furthermore, the fat infiltration with the described "adipogenic composition" in the described ratio allows a significant increase in the initial number of stem cells before differentiation, thus increasing the number of differentiated cells for regenerative and healing processes and tissue engineering and filling. Therefore, in a particularly preferred embodiment of the invention, 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
Therefore, 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 :
1. obtaining a lipoaspirate from liposuction performed by controlled negative pressure syringe;
2. transferring the lipoaspirate into a barrel configured to be subjected to centrifugation and having at the top end a reversible screw capped opening and at the bottom a reversible pressure capped opening;
3. separation of the lipoaspirate fractions by centrifugation at 1500-4500 rpms for 5-15 minutes;
4. assembling the barrel with syringes connected through a luer lock connector to the top opening of the barrel and a cylindrical screw plunger having the outer diameter corresponding to the inner diameter of the barrel at the bottom opening of the barrel;
5. collecting of the fat fractions;
6. mixing stem cells and fat tissue obtained from the relative fractions;
7. optional addition of a composition comprising the association of Insulin like growth factor 1, SH- oligopeptide-2 , calcium chloride to the mixture of the fat and stem cells;
In accordance with another aspect of the present invention it provides a kit for the preparation of autologous adipose tissue for use in aesthetic lipograf procedures comprising:
1 sterile surgical cover cloth 50x50 cm;
3 3 ml syringes with integrated latch lock (luer lock) ;
1 30 ml syringe (luer lock) ;
1 10 ml syringe (luer lock) ;
2 20, 50, 100 ml barrels;
2 screw plungers;
6 luer-lock connectors;
needles or cannulas.
In another embodiment the above described kit further contains:
2 vials containing the lyophilized composition promoting adipocytes proliferation and differentiation;
2 vials containing 1ml of sterile saline.
According to 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.
Further characteristics and advantages deriving from the use of the present invention will clearly appear from the following examples given for indicating and not limiting purposes.
Experimental part
The study was aimed to:
1. demonstrate the presence of adult mesenchymal stem cells in lipoaspirate obtained by the method of the invention;
2. demonstrate in vitro differentiation of adult mesenchymal stem cells and of pre-adipocytes into mature adipocytes under different cell culture conditions ;
3. demonstrate the longer durability of the autologous lipograf obtained by the method in accordance to the invention into three female subj ects .
1. The study has been carried out on 5 subjects (38- 50 years old) . All enrolled patients have given their informed consent to the economic exploitation through patenting of the results of the study and invention that ensued.
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. 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.
According to the International Society for Citotherapy, 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. In this study 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. For the immunohistochemical analysis a specific antibody for beta-integrin 1 was used at a dilution of 1:100 for 1 hour at room temperature. For the detection of antigen-antibody complex 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. In order to verify the ability of the isolated stem cells to differentiate into adult adipose cells and the positive influence of the adipogenic composition in the proliferation and differentiation process, 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) . In panel C Oil Red 0 staining clearly highlighted accumulated lipid droplets in the cells indicating an increased differentiation rate under adipogenic conditions.
3. The longer durability of the autologous lipograf obtained by the method in accordance to the invention into three female subjects is under assessment.