EP3876954A1

EP3876954A1 - A method of obtaining stromal vascular fraction

Info

Publication number: EP3876954A1
Application number: EP20864767.7A
Authority: EP
Inventors: Salih ÖZOVALI; Timur Veysel DO RUOK
Original assignee: T Biyoteknoloji Laboratuvar Estetik Medikal Kozmetik San Tic Ltd Sti
Current assignee: T Biyoteknoloji Laboratuvar Estetik Medikal Kozmetik San Tic Ltd Sti
Priority date: 2019-09-16
Filing date: 2020-04-10
Publication date: 2021-09-15
Also published as: WO2021054909A1; EP3876954A4; TR201913977A1

Abstract

The invention is a method of stromal vascular fraction isolation from fat tissues, comprising the steps of obtaining a sample containing fat tissue taken from the donor; centrifuging the sample until blood, fat, and triglycerides are separated; transferring the fat section into a syringe; coupling the syringe containing the fat with the fat breaking kit syringe from their ends such that the fat is transferred from one syringe to the other syringe through the blades of the fat breaking kit, wherein it is selected for the space between said blades to be 50-2400 microns; repeating the above-mentioned process at least 5 more times by, passing the fat from one syringe to the other through the space between the blades, and repeating this process at least one more time, wherein the blade spaces used in the fat breaking kit are smaller for every repetition; respectively shaking the obtained broken down fat and applying a centrifuge process; collecting the stromal vascular fraction that accumulates at the end of the centrifuge process, at the bottom of the container.

Description

A METHOD OF OBTAINING STROMAL VASCULAR FRACTION

TECHNICAL FIELD

The invention is related to a method for separating/isolating the stromal vascular fraction (SVF) located in the fat cells of mammals, particularly humans, from other components that they are naturally together with.

PRIOR ART

Stem cells are ideal sources to develop regenerative components for the body. The stem cells located in the human body are limited in number, and various protocols are already being developed, in order to exactly optimize the separation/isolation and usage efficiencies of said stem cells.

These stem cells are available not only in the bone marrow in humans but also intensively in the fat cells. Nowadays, stromal vascular fraction (shall be referred to as SVF from now on) containing an abundance of mesenchymal stem cells is obtained from the fat tissues taken from a donor, using various separation/isolation methods. SVF is frequently used in tissue repair and plastic surgery due to its above-mentioned properties.

Besides all of such usages, there are several disadvantages relating to SVF separation/isolation methods. One of the most important disadvantages is that enzyme usage is required. In SVF isolation methods, the fat tissue obtained from the donor needs to be digested with an enzyme, which is generally a collagenase. Following the digestion process, a centrifuge process must be carried out in order to separate the unwanted fat tissues from the SVF that contains the stem cells.

Besides collagenase being an expensive enzyme, it leads to various problems due to usage and obtaining methods. The suitable incubation medium conditions must be obtained to perform collagenase digestion. This medium is provided by using extremely expensive incubation devices that have been customized for said process to be carried out. Besides this, as collagenase is a toxic material for humans, it needs to be separated from the SVF that has been obtained. In order to perform said separation, several numbers of centrifugation, generally 5 centrifugation processes need to be carried out.

When all of these processes are taken into consideration, it can be seen that the total preparation time lasts between 1-1.5 hours.

In the patent document numbered EP3019599B1 a method for isolating SVF has been described. The fat tissue taken from the donor is broken into a single cell suspension by applying a homogenous mechanical force or by mincing and at the same time, SVF is maintained without disrupting its structure. Any kind of collagenase usage is not required during this process.

The quality of the SVF that is obtained is an important factor regarding the success of the process (especially surgical procedures). The term quality mentioned here refers to the regenerative nucleated cell numbers. In the known usage fields, as the number of nucleated cells increases, improvements are observed in the results.

In the method where collagenase is used, the number of nucleated cells varies between 1-1 .5 million/mL in the best results. As it can also be seen from the results in the method described in the patent document numbered EP3019599B1 , this number is approximately 1 million/mL.

In accordance with the explanations provided above, the usage of collagenase and the low numbers of nucleated cells are the most important problems that need to be solved in terms of SVF obtaining methods.

In conclusion, all of these problems mentioned above have made it a necessity to provide a novelty in the prior art.

OBJECTIVE OF THE INVENTION

The present invention aims to overcome the problems mentioned above and to provide a novelty in the prior art.

The main aim of the invention is to convey a method for separating/isolating the stromal vascular fraction (SVF) located in the adipose tissue of mammals, particularly humans, from other components that they are naturally together with. Another aim of the invention is to provide a method that enables cheaper, safer and faster SVF separation/isolation of SVF by eliminating the dependency of using collagenase.

Another aim of the invention is to provide a method which not only eliminates the usage of collagenase but it also ensures to obtain SVF having a higher number of nucleated cells.

BRIEF DESCRIPTION OF THE INVENTION

The present invention that is described above and that can carry out all of the aims to be construed from the detailed description below is a method of stromal vascular fraction isolation from adipose tissue. Accordingly, the method subject to the invention comprises the steps of; obtaining a sample containing adipose tissue taken from a donor; centrifuging the sample until blood, fat and triglycerides are separated; transferring the condensed fat section into a syringe; coupling the syringe containing the fat with the fat breaking kit syringe from their ends such that the fat is transferred from one syringe to the other through the blades of the fat breaking kit, wherein it is selected for the space between said blades to be between 50-2400 microns; repeating the above-mentioned process at least 5 more times, by passing the fat from one syringe to the other through the space between the blades, and repeating this process at least one more time, wherein the blade spaces used in the fat breaking kit are smaller for every repetition; respectively shaking the obtained broken down fat and applying a centrifuge process; collecting of the stromal vascular fraction that accumulates at the bottom of the container at the end of the centrifuge process.

In a preferred embodiment of the invention, said blade spaces are respectively 1200 and 600 microns.

In a preferred embodiment of the invention, the step of transferring fat from one syringe to the other at least 5 times through the space between the blades is repeated two more times and the blade spaces used in the fat breaking kit are selected to be smaller for every repetition. In a preferred embodiment of the invention, said blade spaces are respectively 2400, 1200 and 600 microns.

In a preferred embodiment of the invention, said blade spaces are circular.

In a preferred embodiment of the invention, said blade spaces are pentagonal or hexagonal.

In a preferred embodiment of the invention, flaps have been provided on each side of said spaces such that they are centrifugal.

In a preferred embodiment of the invention, the step of transferring fat from one syringe to the other comprises the process to be repeated at least 9 times where fat is transferred through the space between the blades.

In another preferred embodiment of the invention, the step of transferring fat from one syringe to the other comprises the process to be repeated at least 15 times where fat is transferred through the space between the blades.

In another preferred embodiment of the invention, the step of transferring fat from one syringe to the other comprises the process to be repeated at least 19 times where fat is transferred through the space between the blades.

In another preferred embodiment of the invention, the step of transferring fat from one syringe to the other comprises the process to be repeated at least 25 times where fat is transferred through the space between the blades.

In a preferred embodiment of the invention, the step of transferring fat from one syringe to the other comprises the process to be repeated 31 times where fat is transferred through the space between the blades.

In an another preferred embodiment of the invention, ringer lactate or normal saline is added into the fat whose process of being passed through the blades has been completed.

In a preferred embodiment of the invention, said container is a syringe.

In an another preferred embodiment of the invention, the base part of said syringe is conical. In a preferred embodiment of the invention, the stromal vascular fraction that has accumulated at the base of the container is collected by scraping with a needle. In another preferred embodiment of the invention, the collected stromal vascular fraction is re-suspended.

In a preferred embodiment of the invention, said centrifuge processes are carried out in a swinging bucket centrifuge device.

In a preferred embodiment of the invention, said donor is a mammal. In a preferred embodiment of the invention, said donor is human.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a flow diagram of the method of obtaining stromal vascular fraction (SVF) subject to the invention.

Figure 2 is the illustrative schematic view of the fat breaking and transfer kit which enables transfer from one syringe to the other.

Figure 2A is the view of the blade structure and the detailed view of A section in said structure.

DESCRIPTION OF THE REFERENCE NUMBERS IN THE FIGURES

10. Syringe 11. Breaking kit

12. Blade

121. Blade spaces

122. Flaps

T. Base DETAILED DESCRIPTION OF THE INVENTION

In this detailed description, the method for obtaining stromal vascular fraction subject to the invention is described only to provide a further understanding of the subject matter, without having a limited effect.

The subject matter of the invention is related to a method for separating/isolating the stromal vascular fraction (SVF) located in the fat cells of mammals, particularly humans, from other components that they are naturally together with.

In figure 1 a flow diagram of an alternative embodiment of the method of obtaining stromal vascular fraction (SVF) subject to the invention has been provided. In this figure, the transfer process of fat from one syringe (10) to the other syringe (10) has been shown three times. In the process subject to the invention, two processes of transferring fat from one syringe (10) to the other (10) is also adequate to obtain the desired technical benefit.

The invention is a method of stromal vascular fraction separation from fat tissues, comprising the steps of obtaining a sample containing fat tissue taken from the donor; centrifuging the sample until blood, fat, and triglycerides are separated; transferring the fat section into a syringe (10); coupling the syringe (10) containing the fat with the fat breaking kit (11) syringe (10) from their ends such that the fat is transferred from one syringe (10) to the other syringe (10) through the blades (12) of the fat breaking kit (11 ), wherein it is selected for the space between said blades (121 ) to be between 50-2400 microns; repeating the above-mentioned process at least 5 more times, by passing the fat from one syringe (10) to the other through the space between the blades (121 ), and repeating this process at least one more time, wherein the blade spaces (121 ) used in the fat breaking kit (11 ) are smaller for every repetition; respectively shaking the obtained broken down fat and applying a centrifuge process; collectiing the stromal vascular fraction that accumulates at the end of the centrifuge process, at the bottom of the container.

As used herein the expression “stromal vascular fraction” refers to cell fraction that is derived from the fat tissue vascular component comprising different cells. The fat- derived stem cells can comprise other cells that are immune or that express one or more cells such as mesenchymal cells, hematopoietic cells, hematopoietic stem/progenitor cells, chondrocytes, osteoblasts, osteoclasts, endothelium precursor or progenitor cells, endothelial cells, included but not limited with straight muscles such as pericytes, CD34 + cells (typically found in the umbilical cord), CD29 + cells, CD166 + cells, Thy-1 + or CD90 + stem cells, CD44 + cells, monocytes, leukocytes, lymphocytes, immune cells such as B and T cells, NK cells, macrophages, dendritic cells, neutrophil leukocytes, neutrophils, eosinophils, basophils, granulocytes, erythrocytes, eosinophils, basophils, granulocytes, erythrocytes, megakaryocytes, and thrombocytes. Besides these, said cells can comprise CD3, CD14 (macrophage marker), CD19, CD20 (B cell marker), CD29 (integrin unit), CD31 (endothelial, thrombocytes, macrophage, dendritic cell, granulocyte, T / NK cells, lymphocytes, megakaryocytes, osteoclasts, neutrophils, and others), CD44 (Hyaluronic acid receptor), CD45 (B and T cell marker), CD56, CD73 (lymphocyte differentiation marker) and CD105 markers. It is also possible for the stromal vascular fraction to comprise cells that express any of the markers described in this description and any combination thereof. As used herein, the term “precursor cell” and “stem cell” can be used in substitution for each other and they refer to cells that can transform into different cell types.

Besides this, the term syringe (10) refers to fat syringes (10) that are frequently used in particularly the plastic surgery sector.

The term “donor” refers to living beings that can provide the related fat tissues, preferably to mammals, specifically to humans as mentioned in the detailed description.

In the related method, samples that contain fat tissue are obtained from the donor. Preferably the sample is obtained using a “tumescent technique”. In this technique, fluid is given into the body and the fat cells that are to be extracted together with water are expanded. This fluid also comprises anesthetic inputs. It is unavoidable for the sample obtained using this method to contain a little amount of said fluid.

The samples that are extracted are divided into suitable and preferably equal amounts and are transferred into syringes (10) or suitable container and following this, they are subjected to centrifugation. The aim here is to separate the fat from the remaining sample. The centrifugation process is preferably carried out using a swinging bucket type centrifuge device.

Following centrifugation for a suitable period of time with relative centrifuge force, the sample is separated into three sections as blood, fat, and triglycerides. Besides this, if the sample is obtained using the “tumescent technique”, it is possible for the blood to contain an amount of tumescent fluid.

In a preferred embodiment, the samples obtained are centrifuged for approximately 8 minutes with a relative centrifuge force of 1500 G. The amount of 40cc fat obtained from the donor is transferred into 4 suitable syringes (10) and is centrifuged.

Following centrifugation, blood (or blood-tumescent fluid mixture) is separated such that the blood is located at the bottom section, fat (condensed fat) is located at the midsection, and triglycerides are located at the top section. The fat section located in the middle section is transferred into another syringe without being mixed with the other sections.

A fat breaking kit (11 ) is coupled to the end of the syringe (10) that is filled with the fat section and another syringe (10) end is coupled to the other end of the fat breaking kit (11 ). Said coupling has been given schematically in Figure 2.

An example of suitable fat breaking kits (12) can be the T-Biyoteknoloji Ltd Sti. (T-Lab ®) Microlyzer ®.

Inside said fat breaking kit (12) a blade (12) has been provided that has blade spaces (121 ) such that fat can pass through. An embodiment of the blade structure has been shown in Figure 2. A. Although the blade (12) can have blade spaces (121 ) in different forms, said forms can be selected to have circular, pentagonal, or hexagonal geometry.

In an embodiment of the invention, flaps (122) have been provided on each side of said blade spaces (121) such that they are centrifugal.

The size of said spaces can vary between 50-2400pm, particularly between 600-2400 pm.

The syringe (10) that is full with the fat section is pressed and the fat section is passed through the fat breaking kit (12) and is transferred into the other syringe (10) and following this, the fat is again transferred back from the second syringe (10) into the first syringe (10) by being passed through the fat breaking kit (12). This process is received at least 5 more times. The transfer from one syringe (10) to the other syringe (10) is counted as a process step. The size of said spaces of the blades (12) can vary between 50-2400pm. Preferably the blade spaces can vary between 2400-600 pm. The sizes of the blade spaces (121) are particularly between 1500-600 pm, more specifically between 1200-600 pm. A blade having blade spaces (121 ) of 1200 pm has been used in the application.

The transferring from one syringe (10) to the other (10) can be carried out 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 times.

The situation that must be understood here is that, as the number of transferring one syringe (10) to the other (10) increases, the number of cells also increase and as the number of transfers decrease the amount of SVF that can be obtained at the end of the process also decreases.

The number of the related transfers is preferably between 25-35, however, in the assays carried out, 31 has been determined to be the optimum number.

Following the first fat breaking process, the fat tissue obtained is transferred from a syringe (10) into the fat breaking kit (11 ) as it has been shown in Figure 2, by said fat tissue being passed through another fat breaking kit (11 ). The blade spaces (121) of the blades (12) inside this fat breaking kit (11 ), is smaller from the blade spaces (121 ) that were used during the transfer process from one syringe (10) into the other syringe (10) at the initial stage.

The size of said spaces of the blades (121) can vary between 50-2400pm. Preferably the blade spaces can vary between 2400-600 pm. The sizes of the blade spaces (121 ) are particularly between 300-1500 pm, more specifically between 600-900 pm. A blade having blade spaces (121 ) of 600 pm has been used in the application.

The process of transferring from this second syringe (10) to the other syringe (10) can be carried out 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 times.

The number of the related transfers is preferably between 25-35, however, in the assays carried out, 31 has been determined to be the optimum number. Preferably, the number of transfers from one syringe (10) to the other syringe (10) in this second process is the same as the number of processes carried out in the first process.

In a preferred embodiment of the invention, the step of transferring fat from one syringe (10) to the other at least 5 times through the space between the blades (121 ) must be repeated two more times. This process is carried out three times in total. Here, the sample can be processed through a fat breaking kit (11 ) having a blade (12) with larger blade spaces (121 ) from those mentioned above.

The size of said spaces of the blades can vary between 50-2400pm. Preferably the blade spaces can vary between 2400-50 pm, or they can be particularly 600 pm.

The size of said blade spaces (121 ) mentioned can vary between 50-3000pm. Preferably the blade spaces can vary between 2400-600 pm. The sizes of the blade spaces (121 ) are particularly between 1200-2400 pm, more specifically between 1800-2400 pm. A blade having blade spaces (121 ) of 2400 pm has been used in the application.

The process of transferring from this third syringe (10) to the other syringe (10) can be carried out 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 times.

Preferably, the number of transfers from syringe (10) to the other syringe (10) in this second process is the same as the number of processes carried out in the first process.

Preferably the number of transfers from one syringe (10) to the other syringe (10) are singular numbers. Every time the tissue is passed through the blades (12) the necrotic tissues accumulate on the blade (12) at the initial side. In the process carried out in singular numbers, the fat tissue is finally filled into the other syringe (10) and due to this reason it comprises a very small amount of necrotic tissue or it may not compare any necrotic tissue.

The fat section that is passed from the fat breaking kit (11 ) through the different blade spaces (121 ) three times is collected into a container and preferably ringer lactate or normal saline is added into the container. The ringer lactate or normal saline ratio is approximately 1/8.

The container can be selected as a syringe or a tube.

After ringer lactate or normal saline is added into the container, the container is preferably shaken for 30-60 seconds.

Following this, the container is centrifuged. The centrifuge must be carried out with such relative centrifugal force and time that SVF accumulates at the bottom of the container. In an application, 400 G relative centrifuge force is applied and the desired results have been obtained from the container that was centrifuged for 10 minutes.

In an embodiment of the invention, SVF that sticks to the bottom of the container is collected after being scraped from the related section with a long needle.

In another embodiment of the invention, said container is selected as a syringe and the bottom (T) section of said syringe is conically shaped. The conic geometry mentioned here enables SVF to fill into the end part of the related conical section and therefore it allows easier collection with much lower manual skills in comparison to the SVF’s that stick to the bottom of a normal container.

The SVF that is finally obtained is re-dispersed manually or by vortex and is made ready to be injected.

In the tables provided below, various results showing the cell numbers have been given following the processing of the samples. Cell counting is carried out by the application of a staining dye to SVF that turns the live nucleated cells into green and the dead cells into red and by counting the cell numbers with a suitable device.

The related blood measurements are carried out with a LUNA-STEM model device. Measurement protocols have been arranged as follows;

Dilution factor: 1.11 Minimum cell size: 3 pm Maximum cell size: 3 pm Cell size gating: 3-90 pm Noise reduction: 5 Roundness: 60% Fluorescence threshold: 5 Table 1

As it can be seen, the nucleated cell numbers obtained by this method vary between 3- 8 million. Particularly in example 3, a conical based structure has been used and as a result, the highest number of core cells have been reached.

The protection scope of the invention has been defined in the attached claims, and the invention cannot be limited with the embodiments given as illustration in this detailed description. It is obvious that a person skilled in the art can reach similar embodiments under the light of the description given above without deviating from the main scope of the invention.

Claims

1. The invention is related to a method of stromal vascular fraction isolation from fat tissues; characterized by comprising the steps of; obtaining a sample containing fat tissue taken from a donor; centrifuging the sample until blood, fat and triglycerides are separated; transferring the fat section into a syringe (10); coupling the syringe (10) containing the fat with the fat breaking kit (11 ) syringe (10) from their ends such that the fat is transferred from one syringe (10) to the other syringe (10) through the blades (12) of the fat breaking kit (11 ), wherein it is selected for the space between said blades (121) to be between 50-2400 microns; repeating the above-mentioned process at least 5 more times by, passing the fat from one syringe (10) to the other through the space between the blades (121), and repeating this process at least one more time, wherein the blade spaces (121) used in the fat breaking kit (11 ) are smaller for every repetition; respectively shaking the obtained broken down fat and applying a centrifuge process; collecting the stromal vascular fraction that accumulates at the end of the centrifuge process, at the bottom of the container.

2. A method according to claim 1, characterized in that said blade spaces (121) are respectively 1200 and 600 microns.

3. A method according to claim 1 , characterized in that the process of passing the fat from one syringe (10) to the other through the space between the blades (121 ) at least 5 times is repeated two more times and the blade spaces (121) used in the fat breaking kit (11) are smaller for every repetition.

4. A method according to claim 1 or claim 3, characterized in that said blade spaces (121) are respectively 2400, 1200 and 600 microns.

5. A method according to claim 1, characterized in that said blade spaces (121) are circular.

6. A method according to claim 1, characterized in that said blade spaces (121) are pentagonal or hexagonal.

7. A method according to claim 6, characterized in that flaps (122) have been provided on each side of said blade spaces (121) such that they are centrifugal.

8. A method according to claim 1 , characterized in that the step of transferring fat from one syringe (10) to the other comprises the process to be repeated at least 9 times where fat is transferred through the space (121) between the blades.

9. A method according to claim 1 , characterized in that the step of transferring fat from one syringe (10) to the other comprises the process to be repeated at least 15 times where fat is transferred through the space (121) between the blades.

10. A method according to claim 1 , characterized in that the step of transferring fat from one syringe (10) to the other comprises the process to be repeated at least 19 times where fat is transferred through the space (121) between the blades.

11. A method according to claim 1 , characterized in that the step of transferring fat from one syringe (10) to the other comprises the process to be repeated at least 25 times where fat is transferred through the space (121) between the blades.

12. A method according to claim 1 , characterized in that the step of transferring fat from one syringe (10) to the other comprises the process to be repeated 31 times where fat is transferred through the space (121) between the blades.

13. A method according to claim 1, characterized in that ringer lactate or normal saline is added into the fat whose process of being passed through the blades (12) has been completed.

14. A method according to claim 1 , characterized in that said container is a syringe (10).

15. A method according to claim 14, characterized in that the base (T) part of said syringe (10) is conical.

16. A method according to claim 1, characterized in that the stromal vascular fraction that has accumulated at the base of the container is collected by scraping with a needle.

17. A method according to claim 1, characterized in that the stromal vascular fraction that has been collected is re-resuspended.

18. A method according to claim 1, characterized in that said centrifuge processes are carried out in a swinging bucket centrifuge device.

19. A method according to claim 1, characterized in that said donor is a mammal.

20. A method according to claim 1, characterized in that said donor is human.