WO2017180076A1 - Obtaining stromal vascular cells from human adipose tissue by mechanical separation - Google Patents

Obtaining stromal vascular cells from human adipose tissue by mechanical separation Download PDF

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Publication number
WO2017180076A1
WO2017180076A1 PCT/TR2017/050107 TR2017050107W WO2017180076A1 WO 2017180076 A1 WO2017180076 A1 WO 2017180076A1 TR 2017050107 W TR2017050107 W TR 2017050107W WO 2017180076 A1 WO2017180076 A1 WO 2017180076A1
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Prior art keywords
adipose tissue
disintegration
mechanical separation
disintegrated
vascular cells
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PCT/TR2017/050107
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French (fr)
Inventor
Kemal Tunc Tiryaki
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Kemal Tunc Tiryaki
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Publication of WO2017180076A1 publication Critical patent/WO2017180076A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
    • 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
    • A61M1/89—Suction aspects of liposuction
    • A61M1/892—Suction aspects of liposuction with treatment of the collected fat
    • A61M1/893—Suction aspects of liposuction with treatment of the collected fat with extraction of specific components, e.g. of stem cells
    • 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
    • A61M1/89—Suction aspects of liposuction
    • A61M1/895—Suction aspects of liposuction with means for reinjection of collected fat
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M45/00—Means for pre-treatment of biological substances
    • C12M45/02—Means for pre-treatment of biological substances by mechanical forces; Stirring; Trituration; Comminuting
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M45/00—Means for pre-treatment of biological substances
    • C12M45/05—Means for pre-treatment of biological substances by centrifugation
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
    • C12N2509/10—Mechanical dissociation

Definitions

  • the invention relates to the method of obtaining stem cells that are the main cell types forming the tissues and organs of all multicellular organisms by mechanically.
  • the invention particularly relates to an apparatus for obtaining stem cells mechanically from adipose tissue.
  • the cells that can be converted into any type of cells in an organism and that can continuously be regenerated are called stem cells, meaning the origin of all cells. Their task is to compensate the damages and loss of tissues that may happen in case of injuries, diseases and the like.
  • the perform required repairs by going into the heart in a person who suffers from a heart attack, to the liver in a person who has liver injury, and to the bone line in a person who has a broken bone. They turn into the type of cells or tissues that are needed. The time period they are found most abundant in the body is while the body is in the fetus form.
  • Stem cells are found in all body tissues and organs, and in blood circulation. However, they are more abundant especially in three places. These are the umbilical cord in babies, bone marrow, and the blood circulating in veins.
  • the stem cells circulating in the body can be used in the upcoming years.
  • hormone is given to the patient so as to rapidly proliferate the stem cells found in the bone marrow and pass them into the blood.
  • they are collected from blood with the help of a filter (apheresis) and the remaining blood is given back to the body.
  • This method is still being used.
  • the number of stem cells obtained with this method is less compared to the method of obtaining from bone marrow.
  • tissue augmentation applications have started with mesenchimal stem cells isolated from within the fat (lipoaspirate) taken from the own body of a person. In these applications called enrichment from stem cell, own fat of a patient is taken and injected to the region.
  • tissue regeneration capabilities of the transplanted cells can be made use of, or in other words, they would improve the rejuvenation capability of the skin.
  • Providing the living tissue in a lower amount than the fat injection prevents wrinkles and excessive swelling on face. Moreover, since the cells remain alive in the body, usually a single injection is sufficient. In this technique, it is possible to fill the saggy face parts that became empty underneath (cheeks, upper parts of cheekbones, and around lips), with the fat taken from abdomen or hips.
  • the presently used method of obtaining stem cells takes very long time.
  • stem cells are obtained by collagenase enzyme.
  • use of this collagenase enzyme causes problems in some countries.
  • an application is encountered with application No. EP03759345.6, IPC classification A61 K 38/18, entitled: "Platelet and hematopoietic stem cell production methods" relates to a method comprising application of a TPO mimetic compound to a subject, which leads to increase of HSC production in the subject.
  • the TPO mimetic compound can be applied to a subject alone or together with a pharmaceutically accepted carrier.
  • the TPO mimetic compound can be applied alone or can be combined with one or more additional TPO mimetic compounds and/or other agents improving stem cell mobilization from bone marrow, as well as, for example, G-CSF, SCF, IL-3, and/or Flt-3.
  • Another invention encountered in the literature is the invention No. EP0191 1 175.6, entitled: "multiple-impact stem cells produced from stroma cells obtained from adipose tissue and uses thereof". This invention relates to multiple-impact stem cells produced from stroma cells obtained from adipose tissue and their uses.
  • the invention includes isolated adipose tissue derived stromal cells that have been induced to express at least one phenotypic characteristic of a neuronal, astroglial, hematopoietic progenitor, or hepatic cell.
  • the invention also includes an isolated adipocyte tissue-derived stromal cell that has been dedifferentiated such that there is an absence of adipocyte phenotypic markers.
  • the invention relates to a method for isolation of epithelial or mesenchymal stem/progenitor cells from the amniotic membrane of umbilical cord.
  • the method comprises separating the amniotic membrane from the other components of the umbilical cord in vitro, culturing the amniotic membrane tissue under conditions allowing cell proliferation, and isolating the stem/progenitor cells from the tissue cultures.
  • the present invention relates to the above-mentioned stem cell obtaining method.
  • the main purpose of the invention is to obtain stem cells from own adipose of a person in a quick and simple manner. This is performed by means of an apparatus and improvements made in the process. In this way, doctors can obtain stem cell in a short time period such as 20 minutes in suitable laboratory conditions.
  • Another purpose of the invention is to ensure mechanical digestion/disintegration of the adipose tissue only with the help of cutter knives, without using any organic biological product, enzyme, or medicament.
  • Another purpose of the invention is to enable any doctor to perform suitable operations laboratory conditions to obtain stem cells, without the need for an expert.
  • Another purpose of the invention is to ensure that stem cell obtaining method can performed with lower cost. In this way, everybody can consult stem cell obtaining method.
  • the present invention relates to obtaining stem cells from a person's own fat.
  • Figure - 1. is a general view of the adipose tissue disintegrator apparatus according to the invention.
  • Figure - 2. is a perspective view of the adipose tissue disintegrator apparatus according to the invention.
  • Figure - 3. is a front view of the adipose tissue disintegrator apparatus according to the invention.
  • Figure - 4. is a top view of the adipose tissue disintegrator apparatus according to the invention.
  • Figure - 5. is a detailed view of the adipose tissue disintegrator apparatus according to the invention.
  • Figure - 6. is a general view of the injectors according to the invention.
  • the invention relates to a system (1 ) of obtaining stromal vascular cells from human adipose tissue by mechanical separation, which meets the above said requirements, eliminates all of the drawbacks, and brings some additional advantages.
  • the present invention discloses a system (1 ) of obtaining stromal vascular cells from human adipose tissue by mechanical separation and the structural components of this mechanism, comprising:
  • an adipose tissue disintegrator apparatus comprising an adipose tissue injector section (1 .1 .1 ) enabling disintegration of adipose tissue, a cell knife (1 .1 .2) enabling disintegration of adipose tissue in desired dimensions and easily replaceable due to its grooved surface (1 .1 .2.1 ), and a disintegrated adipose tissue section (1 .1 .3),
  • - injectors (1 .2) consisting of an adipose tissue injector (1 .2.1 ) ensuring placement of the adipose tissue taken from a person via vacuum (liposuction), for disintegration, after it is decanted, centrifuged, and mixed with a buffer solution, and an adipose tissue injector (1 .2.2) sucking the disintegrated adipose tissues found in said disintegrated adipose tissue section (1 .1 .3),
  • the adipose tissue passing through the cell knife (1.1.2) surface is disintegrated and proceeds to the disintegrated adipose tissue section (1.1.3).
  • the disintegrated adipose tissue injector (1.2.2) sucks the disintegrated adipose tissues found in the disintegrated adipose tissue section (1.1.3).
  • FIGS. 1 and Figure 3 show perspective and front views of the adipose tissue disintegration apparatus.
  • the adipose tissue injector section (1.1.1), cell knife (1.1.2), and disintegrated adipose tissue section (1.1.3) are mounted in this order.
  • the cell knife (1.1.2) comprises a disintegrating mechanism (1.1.2.2) such as a wire or knife. Adipose tissue disintegration occurs here.
  • the cell knife (1.1.2) comprises a grooved surface (1.1.2.1). In this way, the cell knife (1.1.2) can be easily coupled with the adipose tissue injector section (1.1.1) and disintegrated adipose tissue section (1.1.3) just like screwing.
  • the disintegration mechanism (1.1.2.2) found on the cell knife (1.1.2) used is found at a different mesh interval. For instance, since the mesh interval of the disintegration mechanism (1.1.2.2) is larger while the tissues that come from the adipose tissue injector (2) to the adipose tissue injection section (1.1.1) pass through the cell knife (1.1.2), the tissues found at the disintegrated adipose tissue section (1.1.3) are larger.
  • this cell knife (1.1.2) grooved surface (1.1.2.1) is demounted from the adipose tissue injector section (1.1.1) and disintegrated adipose tissue section (1.1.3) in a similar fashion to screwing.
  • a cell knife (1.1.2) having a disintegration mechanism (1.1.2.2) with a smaller mesh interval is mounted to the adipose tissue injector section (1.1.1) and disintegrated adipose tissue section (1.1.3) in a fashion similar to screwing.
  • the tissues found at the disintegrated adipose tissue section (1.3) are sucked in by the disintegrated adipose tissue injector (1.3) and re-injected into the adipose tissue injector section (1.1.1). In this way, it is separated into smaller pieces while passing through the disintegration mechanism with smaller mesh interval. This operation can be repeated until the adipose tissue reaches the desired dimensions by replacing with a cell knife (1 .1 .2) having a disintegration mechanism (1 .1 .2.2) with the desired mesh interval.
  • the dimensions of the disintegration mechanism (1 .1 .2.2) of the cell knife (1 .1 .2) can be changed as 3000 micron perforations, 2000 micron perforations, 1000 micron perforations, and 500 micron perforations.
  • the dimensions of the disintegration mechanism (1 .1 .2.2) that forms a part of the cell knife (1 .1 .2) can be changed in the form of perforations with diameters varying from 500 to 3000 microns.
  • the adipose tissue taken from a person via vacuum is placed into a shaker centrifuge used in the known status of the art for separation into layers within injectors used in the known status of the art.
  • the shaker centrifuge used in the known status of the art has 3 fundamental characteristics as follows:
  • Adipose tissue can be centrifuged by automatic setting, c. At the same time, the disintegrated tissue is shaken and mixed by means of intermittent rotation and stopping.
  • the method of obtaining stromal vascular cells from human adipose tissue via mechanical separation comprises:
  • adipose tissue injector (1 .2.1 ) having at least 10 ml adipose tissue and a suitable suction canulla used in the known status of the art
  • the pellet needs to be dissolved in 2-5 cc PBS solution, and if the method is applied for body, then the pellet needs to be dissolved in 5-10 cc PBS solution.

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Abstract

The invention discloses a system (1) of obtaining stromal vascular cells from human adipose tissue by mechanical separation and the structural components of this mechanism, comprising an adipose tissue disintegration apparatus (1.1) comprising an adipose tissue injector section (1.1.1) enabling disintegration of adipose tissue, a cell knife (1.1.2) enabling disintegration of adipose tissue in desired dimensions and easily replaceable due to its grooved surface (1.1.2.1), and a disintegrated adipose tissue section (1.1.3), injectors (1.2) consisting of an adipose tissue injector (1.2.1) ensuring placement of the adipose tissue taken from a person via vacuum (liposuction), for disintegration, before it is decanted, centrifuged, and mixed with a buffer solution, and an adipose tissue injector (1.2.2) sucking the disintegrated adipose tissues found in said disintegrated adipose tissue section (1.1.3), a shaker centrifuge used in the known status of the art for separating the adipose tissue taken from the person via vacuum into layers within injectors used in the known status of the art.

Description

OBTAINING STROMAL VASCULAR CELLS FROM HUMAN ADIPOSE TISSUE BY
MECHANICAL SEPARATION
THE RELATED ART
The invention relates to the method of obtaining stem cells that are the main cell types forming the tissues and organs of all multicellular organisms by mechanically.
The invention particularly relates to an apparatus for obtaining stem cells mechanically from adipose tissue.
THE PRIOR ART
The cells that can be converted into any type of cells in an organism and that can continuously be regenerated are called stem cells, meaning the origin of all cells. Their task is to compensate the damages and loss of tissues that may happen in case of injuries, diseases and the like.
The perform required repairs by going into the heart in a person who suffers from a heart attack, to the liver in a person who has liver injury, and to the bone line in a person who has a broken bone. They turn into the type of cells or tissues that are needed. The time period they are found most abundant in the body is while the body is in the fetus form.
Afterwards, their numbers are reduced with aging. Hence, it is also known that tissue and organ recovery gets slower and becomes harder with aging. Among all the tissues of an adult, the ratio of mature stem cells is only a small percentage of the tissue cells for ensuring integrity and continuity of that tissue.
Stem cells are found in all body tissues and organs, and in blood circulation. However, they are more abundant especially in three places. These are the umbilical cord in babies, bone marrow, and the blood circulating in veins.
It is understood that the stem cells circulating in the body can be used in the upcoming years. For this purpose, firstly, hormone is given to the patient so as to rapidly proliferate the stem cells found in the bone marrow and pass them into the blood. Afterwards, they are collected from blood with the help of a filter (apheresis) and the remaining blood is given back to the body. This method is still being used. However, the number of stem cells obtained with this method is less compared to the method of obtaining from bone marrow. Nowadays, tissue augmentation applications have started with mesenchimal stem cells isolated from within the fat (lipoaspirate) taken from the own body of a person. In these applications called enrichment from stem cell, own fat of a patient is taken and injected to the region. The advantage of enriched tissue injections from stem cell over classical adipose tissue transplants is the reduction in post-transplant loss, since these cells have superior capability of forming new vasculature, and therefore transfer of lower amount of tissues is sufficient. This situation minimizes distention in patients following application. Besides, there are various publications stating that the tissue regeneration capabilities of the transplanted cells can be made use of, or in other words, they would improve the rejuvenation capability of the skin.
Providing the living tissue in a lower amount than the fat injection prevents wrinkles and excessive swelling on face. Moreover, since the cells remain alive in the body, usually a single injection is sufficient. In this technique, it is possible to fill the saggy face parts that became empty underneath (cheeks, upper parts of cheekbones, and around lips), with the fat taken from abdomen or hips. However, there are some problems in the presently used methods of obtaining stem cells. For instance, the presently used method of obtaining stem cells takes very long time.
Also, since the presently used method of obtaining stem cells is expensive, people cannot use this method commonly.
One of the reasons why the presently used method of obtaining stem cells is expensive is because the operation requires expertise. Since the operation cannot be performed by any person, this also causes the operation time to get longer. Moreover, nowadays, stem cells are obtained by collagenase enzyme. However, use of this collagenase enzyme causes problems in some countries. In the literature research, an application is encountered with application No. EP03759345.6, IPC classification A61 K 38/18, entitled: "Platelet and hematopoietic stem cell production methods" relates to a method comprising application of a TPO mimetic compound to a subject, which leads to increase of HSC production in the subject. The TPO mimetic compound can be applied to a subject alone or together with a pharmaceutically accepted carrier. The TPO mimetic compound can be applied alone or can be combined with one or more additional TPO mimetic compounds and/or other agents improving stem cell mobilization from bone marrow, as well as, for example, G-CSF, SCF, IL-3, and/or Flt-3. Another invention encountered in the literature is the invention No. EP0191 1 175.6, entitled: "multiple-impact stem cells produced from stroma cells obtained from adipose tissue and uses thereof". This invention relates to multiple-impact stem cells produced from stroma cells obtained from adipose tissue and their uses. The invention includes isolated adipose tissue derived stromal cells that have been induced to express at least one phenotypic characteristic of a neuronal, astroglial, hematopoietic progenitor, or hepatic cell. The invention also includes an isolated adipocyte tissue-derived stromal cell that has been dedifferentiated such that there is an absence of adipocyte phenotypic markers.
Another invention encountered in the literature is an invention with application No. EP05743325.2, IPC classification C12N 5/073, and entitled "Isolation of stem/progenitor cells from amniotic membrane of umbilical cord". The invention relates to a method for isolation of epithelial or mesenchymal stem/progenitor cells from the amniotic membrane of umbilical cord. The method comprises separating the amniotic membrane from the other components of the umbilical cord in vitro, culturing the amniotic membrane tissue under conditions allowing cell proliferation, and isolating the stem/progenitor cells from the tissue cultures.
When the above given inventions are analyzed, an improvement for solution of the above- mentioned problems in stem cell obtaining method is not encountered. As a result, improvement is to be made in stem cell obtaining methods, and therefore novel embodiments that would eliminate the above said drawbacks and bring solutions to the problems of the prior art systems are needed. BRIEF DESCRIPTION OF THE INVENTION
The present invention relates to the above-mentioned stem cell obtaining method.
The main purpose of the invention is to obtain stem cells from own adipose of a person in a quick and simple manner. This is performed by means of an apparatus and improvements made in the process. In this way, doctors can obtain stem cell in a short time period such as 20 minutes in suitable laboratory conditions.
Another purpose of the invention is to ensure mechanical digestion/disintegration of the adipose tissue only with the help of cutter knives, without using any organic biological product, enzyme, or medicament.
Another purpose of the invention is to enable any doctor to perform suitable operations laboratory conditions to obtain stem cells, without the need for an expert.
Another purpose of the invention is to ensure that stem cell obtaining method can performed with lower cost. In this way, everybody can consult stem cell obtaining method.
In order to achieve the above said purposes which would be understood better with the below given detailed description, the present invention relates to obtaining stem cells from a person's own fat.
For better understanding of the embodiment of the present invention and its advantages with its additional components, it should be evaluated together with below described figures.
BRIEF DESCRIPTION OF THE FIGURES
Figure - 1. is a general view of the adipose tissue disintegrator apparatus according to the invention.
Figure - 2. is a perspective view of the adipose tissue disintegrator apparatus according to the invention.
Figure - 3. is a front view of the adipose tissue disintegrator apparatus according to the invention.
Figure - 4. is a top view of the adipose tissue disintegrator apparatus according to the invention.
Figure - 5. is a detailed view of the adipose tissue disintegrator apparatus according to the invention.
Figure - 6. is a general view of the injectors according to the invention. REFERENCE NUMBERS
1. System of obtaining stromal vascular cells from human adipose tissue by mechanical separation
1.1. Adipose tissue disintegrator apparatus
1.1.1. Adipose tissue injection section
1.1.2. Cell knife
1.1.2.1. Grooved surface
1.1.2.2. Disintegration mechanism
1.1.3. Disintegrated adipose tissue section
1.2. Injector
1.2.1. Adipose tissue injector
1.2.2. Disintegrated adipose tissue injector
DETAILED DESCRIPTION OF THE INVENTION
The invention relates to a system (1 ) of obtaining stromal vascular cells from human adipose tissue by mechanical separation, which meets the above said requirements, eliminates all of the drawbacks, and brings some additional advantages.
In this detailed description, the novelty according to the invention is only disclosed for better understanding of the subject without forming any limiting effect. Accordingly, in the following detailed description and figures, the present invention discloses a system (1 ) of obtaining stromal vascular cells from human adipose tissue by mechanical separation and the structural components of this mechanism, comprising:
- an adipose tissue disintegrator apparatus (1 .1 ) comprising an adipose tissue injector section (1 .1 .1 ) enabling disintegration of adipose tissue, a cell knife (1 .1 .2) enabling disintegration of adipose tissue in desired dimensions and easily replaceable due to its grooved surface (1 .1 .2.1 ), and a disintegrated adipose tissue section (1 .1 .3),
- injectors (1 .2) consisting of an adipose tissue injector (1 .2.1 ) ensuring placement of the adipose tissue taken from a person via vacuum (liposuction), for disintegration, after it is decanted, centrifuged, and mixed with a buffer solution, and an adipose tissue injector (1 .2.2) sucking the disintegrated adipose tissues found in said disintegrated adipose tissue section (1 .1 .3),
- a shaker centrifuge used in the known status of the art for separating the adipose tissue taken from the person via vacuum into layers within injectors used in the known status of the art. As can be seen in Figure 1, after the adipose tissue taken from a person via vacuum (liposuction) is decanted, centrifuged, and mixed with a buffer solution, it is sucked into the adipose tissue injector (1.2.1) for disintegration. Afterwards, the adipose tissue found in the adipose tissue injector (1.2.1) is transferred to the adipose tissue injector section (1.1.1). The adipose tissue pushed by pressure from the adipose tissue injector (1.2.1 ) shown in Figure 6, proceeds quickly to the cell knife (1.1.2) from the adipose tissue injector section (1.1.1). Here, the adipose tissue passing through the cell knife (1.1.2) surface is disintegrated and proceeds to the disintegrated adipose tissue section (1.1.3). The disintegrated adipose tissue injector (1.2.2) sucks the disintegrated adipose tissues found in the disintegrated adipose tissue section (1.1.3).
Figure 2 and Figure 3 show perspective and front views of the adipose tissue disintegration apparatus. As can be seen from the figures, the adipose tissue injector section (1.1.1), cell knife (1.1.2), and disintegrated adipose tissue section (1.1.3) are mounted in this order.
As can be seen from Figure 4, the cell knife (1.1.2) comprises a disintegrating mechanism (1.1.2.2) such as a wire or knife. Adipose tissue disintegration occurs here. As can be seen from Figure 5, the cell knife (1.1.2) comprises a grooved surface (1.1.2.1). In this way, the cell knife (1.1.2) can be easily coupled with the adipose tissue injector section (1.1.1) and disintegrated adipose tissue section (1.1.3) just like screwing.
In order to perform adipose tissue disintegrating operation, more than one cell knives (1.1.2) are used. The disintegration mechanism (1.1.2.2) found on the cell knife (1.1.2) used is found at a different mesh interval. For instance, since the mesh interval of the disintegration mechanism (1.1.2.2) is larger while the tissues that come from the adipose tissue injector (2) to the adipose tissue injection section (1.1.1) pass through the cell knife (1.1.2), the tissues found at the disintegrated adipose tissue section (1.1.3) are larger.
Afterwards, this cell knife (1.1.2) grooved surface (1.1.2.1) is demounted from the adipose tissue injector section (1.1.1) and disintegrated adipose tissue section (1.1.3) in a similar fashion to screwing. In preparation for the next stage, a cell knife (1.1.2) having a disintegration mechanism (1.1.2.2) with a smaller mesh interval is mounted to the adipose tissue injector section (1.1.1) and disintegrated adipose tissue section (1.1.3) in a fashion similar to screwing. The tissues found at the disintegrated adipose tissue section (1.3) are sucked in by the disintegrated adipose tissue injector (1.3) and re-injected into the adipose tissue injector section (1.1.1). In this way, it is separated into smaller pieces while passing through the disintegration mechanism with smaller mesh interval. This operation can be repeated until the adipose tissue reaches the desired dimensions by replacing with a cell knife (1 .1 .2) having a disintegration mechanism (1 .1 .2.2) with the desired mesh interval. Briefly, the dimensions of the disintegration mechanism (1 .1 .2.2) of the cell knife (1 .1 .2) can be changed as 3000 micron perforations, 2000 micron perforations, 1000 micron perforations, and 500 micron perforations. In other words, the dimensions of the disintegration mechanism (1 .1 .2.2) that forms a part of the cell knife (1 .1 .2) can be changed in the form of perforations with diameters varying from 500 to 3000 microns.
The adipose tissue taken from a person via vacuum is placed into a shaker centrifuge used in the known status of the art for separation into layers within injectors used in the known status of the art. The shaker centrifuge used in the known status of the art has 3 fundamental characteristics as follows:
a. It minimizes the contact of the collected oil with air and it is designed with a 50 cm rotating boiler diameter such that it would accommodate the 50 cc injectors used in the known status of the art, in order to simplify the process, b. Adipose tissue can be centrifuged by automatic setting, c. At the same time, the disintegrated tissue is shaken and mixed by means of intermittent rotation and stopping. The method of obtaining stromal vascular cells from human adipose tissue via mechanical separation comprises:
- collection of fat by vacuum, using an adipose tissue injector (1 .2.1 ) having at least 10 ml adipose tissue and a suitable suction canulla used in the known status of the art,
- medium level disintegration of the adipose tissue found in the adipose tissue injector (1 .2.1 ) by passing it through a cell knife (1 .1 .2) with a perforation diameter of 3000 microns, for at least 1 minute,
- afterwards, disintegration of the adipose tissue into smaller pieces compared to the previously disintegrated adipose tissue pieces, by means of passing the adipose tissue through a cell knife (1 .1 .2) with a perforation diameter of 500-1000 microns, - adding 1 ml PBS solution (buffer solution) to each 5 cc adipose tissue and stirring,
- leaving the mechanically disintegrated adipose tissue to incubation in PBS solution for 15-60 minutes,
- centrifuging the incubated tissue for 5-10 minutes, following centrifugation, removal of the supernatant, or in other words, the low density liquid that stays on the top, without damaging the cellular pellet, or in other words, the high density liquid that stays at the bottom, dissolving the pellet in PBS and making it ready for administration to the patient.
If the method of obtaining stromal vascular cells from human adipose tissue by mechanical separation is applied for face, then the pellet needs to be dissolved in 2-5 cc PBS solution, and if the method is applied for body, then the pellet needs to be dissolved in 5-10 cc PBS solution.

Claims

1. A method of obtaining stromal vascular cells from human adipose tissue by mechanical separation, and it is characterized in comprising the operation steps of:
- collection of fat by vacuum, using an adipose tissue injector (1 .2.1 ) having at least 10 ml adipose tissue and a suitable suction canulla used in the known status of the art,
- disintegration of the adipose tissue found in the adipose tissue injector (1 .2.1 ) by passing it through the cell knife (1 .1 .2) for at least 1 minute,
- afterwards, disintegration of the adipose tissue into smaller pieces compared to the previously disintegrated adipose tissue pieces, by means of passing the adipose tissue through the cell knife (1 .1 .2) again,
- disintegration into smaller pieces,
- adding PBS solution (buffer solution) onto adipose tissue and mixing,
- leaving the mechanically disintegrated adipose tissue to incubation in PBS solution,
- centrifugation of the incubated tissue,
- following centrifugation, removal of the supernatant, or in other words, the low density liquid that stays on the top, without damaging the cellular pellet, or in other words, the high density liquid that stays at the bottom,
- dissolving the pellet in PBS solution and making it ready for administration to the patient.
The method of obtaining stromal vascular cells from human adipose tissue by mechanical separation according to claim 1 , and characterized in that; it comprises adding at least 1 ml PBS solution (buffer solution) to each 5 cc adipose tissue and stirring. The method of obtaining stromal vascular cells from human adipose tissue by mechanical separation according to claim 1 , and characterized in that; it comprises leaving the mechanically disintegrated adipose tissue to incubation in PBS solution for 15-60 minutes.
4. The method of obtaining stromal vascular cells from human adipose tissue by mechanical separation according to claim 1 , and characterized in that; it comprises centrifuging the incubated tissue for 5-10 minutes. 5. The method of obtaining stromal vascular cells from human adipose tissue by mechanical separation according to claim 1 , and characterized in that; if the method is applied for face, the pellet is dissolved in 2-5 cc PBS solution.
6. The method of obtaining stromal vascular cells from human adipose tissue by mechanical separation according to claim 1 , and characterized in that; if the method is applied for body, the pellet is dissolved in 5-10 cc PBS solution.
7. The method of obtaining stromal vascular cells from human adipose tissue by mechanical separation according to claim 1 , and characterized in that; it comprises disintegration of the adipose tissue found in the adipose tissue injector (1 .2.1 ) by passing it through a cell knife (1 .1 .2) with a perforation diameter of 3000 microns, for at least 1 minute.
8. The method of obtaining stromal vascular cells from human adipose tissue by mechanical separation according to claim 1 , and characterized in that; it comprises disintegration of the adipose tissue into smaller pieces compared to the previously disintegrated adipose tissue pieces, by means of passing the adipose tissue through a cell knife (1 .1 .2) with a perforation diameter of 500-1000 microns.
9. A system of obtaining stromal vascular cells from human adipose tissue by mechanical separation, and characterized in comprising:
- an adipose tissue disintegration apparatus (1 .1 ) allowing disintegration of the adipose tissue,
- injectors (1 .2) injecting the adipose tissue taken from a person via vacuum (liposuction) to the adipose tissue injection section (1 .1 .1 ) for disintegration, after it is decanted, centrifuged, and mixed with a buffer solution, and sucking the disintegrated adipose tissues found in the disintegrated adipose tissue section (1 .1 .3).
10. The system of obtaining stromal vascular cells from human adipose tissue by mechanical separation according to claim 9, and characterized in that; it comprises the adipose tissue injector section (1 .1 .1 ), which is the section of the adipose tissue disintegration apparatus (1 .1 ), and which ensures disintegration of adipose tissue.
11. The system of obtaining stromal vascular cells from human adipose tissue by mechanical separation according to claim 9, and characterized in that; it comprises the cell knife (1 .1 .2) enabling disintegration of adipose tissue in desired dimensions and can be replaced easily.
12. The system of obtaining stromal vascular cells from human adipose tissue by mechanical separation according to claims 9 to 1 1 , and characterized in that; it comprises a disintegration mechanism (1 .1 .2.2) that forms a part of the cell knife (1 .1 .2) and has perforations with diameters varying from 500 to 3000 microns.
13. The system of obtaining stromal vascular cells from human adipose tissue by mechanical separation according to claims 9 to 1 1 , and characterized in that; it comprises a grooved surface (1 .1 .2.1 ), which is a part of the cell knife (1 .1 .2), and which can be mounted to and demounted from the adipose tissue injector section (1 .1 .1 ) and disintegrated adipose tissue section (1 .1 .3) in a way similar to screwing.
14. The system of obtaining stromal vascular cells from human adipose tissue by mechanical separation according to claim 9, and characterized in that; it comprises the disintegrated adipose tissue section (1 .1 .3) which is the part where the adipose tissue passing through the cell knife (1 .1 .2) reaches after being disintegrated.
15. The system of obtaining stromal vascular cells from human adipose tissue by mechanical separation according to claim 9, and characterized in that; it comprises the adipose tissue injector (1 .2.1 ), which is one of the parts of the injectors (1 .2), injecting the adipose tissue taken from a person via vacuum (liposuction) to the adipose tissue injection section (1 .1 .1 ) for disintegration, after it is decanted, centrifuged, and mixed with a buffer solution.
16. The system of obtaining stromal vascular cells from human adipose tissue by mechanical separation according to claim 9, and characterized in that; it comprises the disintegrated adipose tissue injector (1 .2.2) which sucks the disintegrated adipose tissues found in the disintegrated adipose tissue section (1 .1 .3).
PCT/TR2017/050107 2016-04-13 2017-03-21 Obtaining stromal vascular cells from human adipose tissue by mechanical separation WO2017180076A1 (en)

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