WO2018160096A1 - Device for fractionating adipose tissue and extracting the stromal vascular fraction therefrom for use in regenerative medicine - Google Patents
Device for fractionating adipose tissue and extracting the stromal vascular fraction therefrom for use in regenerative medicine Download PDFInfo
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- WO2018160096A1 WO2018160096A1 PCT/RU2017/050097 RU2017050097W WO2018160096A1 WO 2018160096 A1 WO2018160096 A1 WO 2018160096A1 RU 2017050097 W RU2017050097 W RU 2017050097W WO 2018160096 A1 WO2018160096 A1 WO 2018160096A1
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Classifications
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- 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
- C12M1/00—Apparatus for enzymology or microbiology
- C12M1/12—Apparatus for enzymology or microbiology with sterilisation, filtration or dialysis means
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- 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
- C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
- C12M3/06—Tissue, human, animal or plant cell, or virus culture apparatus with filtration, ultrafiltration, inverse osmosis or dialysis means
Definitions
- the proposed device relates to medicine and medical biotechnology, namely to regenerative medicine and cell technology and can be used to treat adipose tissue obtained by liposuction, for further use as a source of mesenchymal stromal cells, stromal vascular fraction of adipose tissue, for lipofilling or cryopreservation .
- Intact adipose tissue is a tissue rich in blood vessels, consisting of mature fat cells (adipocytes), a stromal-vascular cell fraction and supporting stroma.
- the stromal-vascular cell fraction is a cell complex that includes adipose tissue stem cells (SCL), endothelial and smooth muscle cells of blood vessels and their predecessors, pericytes, fibroblasts, red blood cells - red blood cells and white blood cells.
- SVF adipose tissue stem cells
- endothelial and smooth muscle cells of blood vessels and their predecessors pericytes, fibroblasts, red blood cells - red blood cells and white blood cells.
- the main component of SVF are adipose tissue stem cells capable of self-renewal and multipotent differentiation. Due to their plasticity, they are considered the most promising objects for cell therapy, find their application in the treatment of various injuries and diseases.
- the clinical use of the stromal-vascular fraction includes the regeneration of soft tissues and bones, cosmetic defects, chronic trophic and radiation ulcers, burns, Crohn's disease, multiple sclerosis, in the graft versus host reaction, in myocardial infarction and strokes of various origins.
- a known method of isolating stem cells from adipose tissue using the manual method (Zuk PA, Zhu M., Mizuno N. et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng. 2001; 7 (2): 21 1 -28.).
- This method is based on the treatment of lipoaspirate with a 0.075% type I collagenase solution at 37 ° C for 30 minutes and subsequent centrifugation of the resulting suspension at 800 d.
- the cell suspension After centrifugation, the cell suspension is divided into two fractions: adipocytes are in the upper layer and the sediment is in the sediment - stromal-vascular fraction with an admixture of red blood cells, which were removed during incubation in a lysis solution of ammonium chloride.
- the disadvantages of this method are the high time and organizational costs, the presence of the "human factor”, a high risk of impaired sterility of the process and contamination of biological material and the final product.
- a device is known from the prior art (System for processing lipoaspirate cells EP 1921 133 A2, CYTORI THERAPEUTICS, INC (US), IPC A61 K31 / 436, published May 14, 2008), which is a closed system for processing lipoaspirate obtained during liposuction .
- the system connects directly to the lipoaspiration cannula. It includes a vacuum pump, a container for collecting lipoaspirate, a mixer for mixing the processed biomaterial with additives and activators, a system of two filters with pores of different diameters.
- the first filter divides the lipoaspirate into 2 fractions, where one fraction contains a population of cells, including stem cells of adipose tissue, and the other fraction contains lipids, blood, adipocytes and saline.
- the first filter rotates and structurally divides the capacity into two chambers, for the corresponding fractions, and a second filter concentrates the cell fraction before being fed to the mixer.
- the conductive line allows you to extract the final fraction without violating the tightness of the system and direct it to a centrifuge, then extract the obtained fraction aseptically.
- the device allows you to enter activators and additives.
- the system also has a built-in temperature controller.
- the main disadvantage of this device is that at the first stage of processing, the lipoaspirate is filtered without providing destruction of the stroma of the tissue, which fixes the cells of the stromal-vascular fraction and does not allow them to pass through the filter, thereby reducing the concentration of cells in the permeate. Also, the shortcomings of the system are long trunk lines and a significant number of junctions, which are an environment for adhesion and loss of target cells.
- a device is known from the prior art (Device for separating adult stem cell US 20130344589 A1, HUMAN MED AG (DE), PC C12M1 / 00, publ. 12/26/2003), which is a system comprising an adipose tissue container equipped with a feeding device liquids for washing biomaterial, a mixture of necessary reagents, alternately with extracting the fraction containing stem cells, a valve for equalizing pressure, a piston, a vibrator, a semi-permeable membrane having an electrostatic charge and a valve that divide the container into 2 parts, with a temperature controller tours.
- One of the chambers is equipped with a device for mixing the initial biomaterial with additives, performing rotational and / or pendulum movements.
- the main disadvantage of this device is the method of creating pressure due to the piston, which leads to a high mechanical load on the target cells due to punching through the filter and edge destruction of the cells at the junction of the piston to the cylinder wall.
- a device for the selection of adipose stem cells (System and methods for preparation of adipose-derived stem cells, US 20130012921 A1, PUSTILNIK FELIX, PC A61 M37 / 00, publ. 10.01 .2013), which is a cone-shaped container, is hermetically sealed, as a prototype a closed lid equipped with connectors for the introduction and removal of biomaterial and liquids for its processing, as well as an auxiliary tube for concentration, the resulting stromal-vascular fraction.
- the disadvantages of this system are the presence of two test tubes, which increases the risk of contamination of the material, its loss when transferred between containers, entails additional requirements for auxiliary equipment.
- the technical task of the present invention is to increase the efficiency of isolation of the stromal-vascular fraction while maintaining maximum cell viability and maintaining its regenerative potential.
- a general technical result of the invention is the isolation of the stromal-vascular fraction of adipose tissue, characterized by a high concentration and viability of cells, the absence of debris and residues of the stroma and adipose tissue.
- the technical problem is achieved through the use of a sealed device consisting of two chambers located one above the other and separated by at least one strainer, both chambers being in one housing, and the lower chamber with a volume of 3 - 9 ml has a keeled shape, is located at an angle 10 - 45 degrees and extended along the entire length of the device.
- the proposed device is a closed sealed system in the form of a transparent cone-shaped container with a lid with a volume of 300-500 ml, with an external graduation in volume and a system of internal parietal canals, the container being separated by at least one strainer adjacent flush to the interface of the chambers , into two chambers - a working chamber for processing tissue with a parabolic bottom without acute angles in the working space and a chamber for concentrating cells, moreover, a chamber for concentrating cells has t the volume of 3 - 9 ml, has a keeled shape, is located at an angle of 10 - 45 degrees and stretched along the entire length of the device.
- the device has at least 6 isolated channels located on the side walls of the tank on the inside, and the input of each channel has a mount for Luer-Lock type adapters.
- the device has a channel for introducing biological material, ending in a working chamber for processing tissue.
- the device has a channel for introducing reagents and wash buffers, ending in a working chamber for processing tissue.
- the device has at least 3 channels for sampling the supernatant, with one ending in the working chamber at the attachment point of the strainer, and the other two ending in 1/3 - 1/2 of the length of the inner chamber.
- the device has a channel for selecting the final product — the stromal-vascular fraction of adipose tissue, ending at the base of the container in the cell concentration chamber, the channel exit being located at the sharp end of the keel of the cell concentration chamber.
- the device may have additional channels for introducing or selecting components ending in a working chamber for processing tissue and / or in a chamber for concentrating cells.
- the device has a bacteriological filter for balancing the pressure inside the system, and the bacteriological filter is located on the cover of the device.
- the biological tissue placed in the device is washed from the remnants of the circulating blood; it is subjected to enzymatic treatment, due to which the stromal tissue is lysed and the stromal-vascular fraction enters the medium, the cell component and stromal and adipose tissue residues are separated by centrifugation through a microfilter, followed by washing of the cell fraction from residual enzymes and its concentration. Due to the parabolic bottom without sharp corners, the cell processing chamber and the keeled shape of the cell concentration chamber increase cell yield. Due to the location of the chamber for concentrating cells at an angle and along the entire length of the device, the risk of the appearance of “dead” zones is minimized and the yield of cells is increased.
- the presence of input / output channels with outputs at different levels inside the system wall allows minimizing the total area of the internal surface of the system, which reduces the risk of cell adhesion on the surface of the tubes; It allows the controlled wall-wise introduction of biological material and reagents, which reduces the physical effect on the biological material and increases the degree of cell survival; allows you to fully select the supernatant with the remains of the stroma of adipose tissue, without affecting the sediment; It allows you to completely select the sediment in the form of a stromal-vascular fraction of adipose tissue.
- FIG. 1 - shows a three-dimensional General view of a device for fractionation of adipose tissue and allocation of stromal-vascular fraction
- FIG. 2 shows a device for fractioning adipose tissue and isolating a stromal-vascular fraction in a section.
- figure 1 and figure 2 shows a device for fractionation of adipose tissue and isolation of the stromal-vascular fraction, which is a sealed container (A) with a lid (B), divided by a mesh microfilter (C) into two chambers - a tissue processing chamber (D) and a cell concentration chamber (D).
- the device has a system of internal parietal channels: for the introduction of lipoaspirate (1), for the introduction of reagents and buffers (2), for the selection of supernatant and excess fluid (3-5), for the selection of the stromal-vascular layer (6).
- the operation of the device is a process of stepwise washing and treating adipose tissue with proteolytic enzymes in order to isolate the stromal-vascular fraction, where adipose tissue - lipoaspirate is introduced into the device - a sealed container (A), namely, into the tissue processing chamber (D) through channel (1).
- adipose tissue - lipoaspirate is introduced into the device - a sealed container (A), namely, into the tissue processing chamber (D) through channel (1).
- Biological tissue is washed from the remnants of blood in buffer solution introduced through the channel (2). Excess fluid is removed from the system through a channel (3-5).
- Biological tissue is subjected to enzymatic treatment with a mixture of proteolytic enzymes introduced through the channel (2).
- the device is subjected to centrifugation in which the stromal-vascular fraction passes through a microfilter (B) into the cell concentration chamber (D). Excess liquid is removed through the channel (3-5), the precipitate
- the stromal-vascular fraction was isolated from three lipoaspirate samples obtained from healthy adult donors who signed a voluntary informed consent. Fat sampling was carried out according to the standard technique of syringe tuminescent liposuction under local infiltration anesthesia in the region of the anterior abdominal wall.
- the lipoaspirate was washed with a Hartman solution, bringing its volume to 400 ml in a flask. 5 minutes after adding the Hartman solution, all the liquid part was removed by syringe through the port for collecting SVF. The remaining volume of adipose tissue was measured using a measuring scale on the flask. Enzymatic digestion was performed by adding an equivalent volume of NB-6 collagenase solution (GMP Grade, SERVA Electrophoresis GmbH, 0.3 PZ / ml), at 37 ° C for 30 minutes with constant stirring. After that, the flask was centrifuged with ZOOd, 10 min, 25 ° ⁇ .
- NB-6 collagenase solution GMP Grade, SERVA Electrophoresis GmbH, 0.3 PZ / ml
- a 1 ml aliquot of SVF was used to count the number of nucleated cells using a hemacytometer.
- adipose tissue including
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Abstract
The proposed device relates to medical biotechnology and cell technology, and is designed to fractionate adipose tissue and extract the stromal vascular fraction therefrom for purposes of regenerative medicine. The technical result of the invention is controlled extraction of the stromal vascular fraction of adipose tissue, said extraction being characterized by a high cell survival rate, by an absence of debris, remnants of stromal or fatty tissue, or circulating blood cells, and by low concentration and activity of residual enzymes. The technical aim is achieved by using a sealed device consisting of two chambers positioned one on top of the other and separated by at least one mesh filter, wherein the lower chamber is keel-shaped, positioned at an angle and extends along the entire length of the device.
Description
УСТРОЙСТВО ДЛЯ ФРАКЦИОНИРОВАНИЯ ЖИРОВОЙ ТКАНИ И ВЫДЕЛЕНИЯ ИЗ НЕЕ СТРОМАЛЬНО-ВАСКУЛЯРНОЙ ФРАКЦИИ ДЛЯ ПРИМЕНЕНИЯ В DEVICE FOR FACTION OF FAT TISSUE AND ISOLATION FROM HER STROMAL-VASCULAR FACTION FOR APPLICATION IN
РЕГЕНЕРАТИВНОЙ МЕДИЦИНЕ REGENERATIVE MEDICINE
ОБЛАСТЬ ТЕХНИКИ FIELD OF TECHNOLOGY
Предложенное устройство относится к медицине и медицинской биотехнологии, а именно к регенеративной медицине и клеточной технологии и может быть использовано для обработки жировой ткани, полученной при липосакции, для дальнейшего использования в качестве источника мезенхимальных стромальных клеток, стромальной васкулярной фракции жировой ткани, для липофилинга или криоконсервирования. The proposed device relates to medicine and medical biotechnology, namely to regenerative medicine and cell technology and can be used to treat adipose tissue obtained by liposuction, for further use as a source of mesenchymal stromal cells, stromal vascular fraction of adipose tissue, for lipofilling or cryopreservation .
УРОВЕНЬ ТЕХНИКИ BACKGROUND
Интактная жировая ткань представляет собой богатую кровеносными сосудами ткань, состоящую из зрелых жировых клеток (адипоцитов), стромально- васкулярной клеточной фракции и поддерживающей стромы. Intact adipose tissue is a tissue rich in blood vessels, consisting of mature fat cells (adipocytes), a stromal-vascular cell fraction and supporting stroma.
Стромально-васкулярная клеточная фракция (СВФ) - это клеточный комплекс, включающий в себя стволовые клетки жировой ткани (СКЖТ), эндотелиальные и гладкомышечные клетки кровеносных сосудов и их предшественники, перициты, фибробласты, клетки крови - эритроциты и лейкоциты. Основным компонентом СВФ являются стволовые клетки жировой ткани, способные к самообновлению и мультипотентной дифференцировке. Благодаря их пластичности, они считаются наиболее перспективными объектами для клеточной терапии, находят свое применение при лечении различных травм и заболеваний. The stromal-vascular cell fraction (SVF) is a cell complex that includes adipose tissue stem cells (SCL), endothelial and smooth muscle cells of blood vessels and their predecessors, pericytes, fibroblasts, red blood cells - red blood cells and white blood cells. The main component of SVF are adipose tissue stem cells capable of self-renewal and multipotent differentiation. Due to their plasticity, they are considered the most promising objects for cell therapy, find their application in the treatment of various injuries and diseases.
Впервые выделение стромально-васкулярной фракции жировой ткани было описано в 2001 г. году в журнале «Tissue Engineering* Zuk с соавт. Для выделения СВФ авторы использовали ферментативную обработку с последующей отмывкой и осаждением центрифугированием ядросодержащих клеток. СВФ изначально использовалась для получения чистой популяции СКЖТ путем культивирования. The first isolation of the stromal-vascular fraction of adipose tissue was described in 2001 in the journal Tissue Engineering * Zuk et al. To isolate SVF, the authors used enzymatic treatment followed by washing and sedimentation by centrifugation of nucleated cells. SVF was originally used to obtain a pure population of SCLC by cultivation.
С этого времени были проведены многочисленные исследования возможностей клинического применения SVF и ADSCs, показавшие значительные
перспективы использования этих клеток в клинической практике. Клиническое применение стромально-васкулярной фракции включает регенерацию мягких тканей и костей, косметические дефекты, хронические трофические и лучевые язвы, ожоги, болезнь Крона, рассеянный склероз, при реакции трансплантат против хозяина, при инфаркте миокарда и инсультах различного генеза. Since that time, numerous studies have been conducted on the clinical feasibility of SVF and ADSCs, showing significant Prospects for the use of these cells in clinical practice. The clinical use of the stromal-vascular fraction includes the regeneration of soft tissues and bones, cosmetic defects, chronic trophic and radiation ulcers, burns, Crohn's disease, multiple sclerosis, in the graft versus host reaction, in myocardial infarction and strokes of various origins.
В то же время существует целый ряд ограничивающих факторов и, в первую очередь, связанных с разработкой эффективной и безопасной технологии выделения и изоляции клеточного материала. At the same time, there are a number of limiting factors, primarily related to the development of an effective and safe technology for the isolation and isolation of cellular material.
Известен способ выделения стволовых клеток из жировой ткани с использованием ручного метода (Zuk P. A., Zhu М., Mizuno Н. et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng. 2001 ; 7(2): 21 1 -28.). Данный метод основан на обработке липоаспирата 0,075% раствором коллагеназы I типа при температуре 37°С в течение 30 минут и последующим центрифугированием получившейся суспензии при 800 д. После центрифугирования суспензия клеток разделяется на две фракции: в верхнем слое - супернатанте находятся адипоциты, а в осадке - стромально-васкулярная фракция с примесью эритроцитов, которые удаляли во время инкубации в лизирующем растворе хлорида аммония. Недостатками данного метода являются высокие временные и организационные затраты, наличие «человеческого фактора», высокий риск нарушения стерильности процесса и контаминации биологического материала и конечного продукта. A known method of isolating stem cells from adipose tissue using the manual method (Zuk PA, Zhu M., Mizuno N. et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng. 2001; 7 (2): 21 1 -28.). This method is based on the treatment of lipoaspirate with a 0.075% type I collagenase solution at 37 ° C for 30 minutes and subsequent centrifugation of the resulting suspension at 800 d. After centrifugation, the cell suspension is divided into two fractions: adipocytes are in the upper layer and the sediment is in the sediment - stromal-vascular fraction with an admixture of red blood cells, which were removed during incubation in a lysis solution of ammonium chloride. The disadvantages of this method are the high time and organizational costs, the presence of the "human factor", a high risk of impaired sterility of the process and contamination of biological material and the final product.
Из предшествующего уровня техники известно устройство (System for processing lipoaspirate cells ЕР 1921 133 A2, CYTORI THERAPEUTICS, INC (US), МПК A61 K31 /436, опубл. 14.05.2008), представляющее собой замкнутую систему для обработки липоаспирата, полученного в ходе липосакции. Система подсоединяется непосредственно к липоаспирационной канюле. Включает вакуумный насос, контейнер для сбора липоаспирата, миксер для смешивания обработанного биоматериала с добавками и активаторами, систему из двух фильтров с порами разного диаметра. Первый фильтр разделяет липоаспират на 2 фракции, где одна фракция содержит популяцию клеток, включающую в себя стволовые клетки жировой ткани, а другая фракция содержит липиды, кровь, адипоциты и солевой раствор. При этом первый фильтр вращается и конструктивно делит ёмкость на две камеры, для соответствующих фракций, а
второй фильтр концентрирует клеточную фракцию перед подачей в миксер. Проводящая магистраль позволяет извлечь конечную фракцию, не нарушая герметичности системы и направить её в центрифугу, затем асептично извлечь полученную фракцию. Устройство позволяет вводить активаторы и добавки. Также система имеет встроенный контроллер температуры. Основным недостатком данного устройства является то, что на первом этапе обработки липоаспират фильтруется без обеспечения разрушения стромы ткани, которая фиксирует клетки стромально-васкулярной фракции и не позволяет им пройти через фильтр, снижая тем, самым концентрацию клеток в пермеате. Также недостатками системы являются магистрали большой протяженности и значительное количество примыканий, являющиеся средой для адгезии и потери целевых клеток. A device is known from the prior art (System for processing lipoaspirate cells EP 1921 133 A2, CYTORI THERAPEUTICS, INC (US), IPC A61 K31 / 436, published May 14, 2008), which is a closed system for processing lipoaspirate obtained during liposuction . The system connects directly to the lipoaspiration cannula. It includes a vacuum pump, a container for collecting lipoaspirate, a mixer for mixing the processed biomaterial with additives and activators, a system of two filters with pores of different diameters. The first filter divides the lipoaspirate into 2 fractions, where one fraction contains a population of cells, including stem cells of adipose tissue, and the other fraction contains lipids, blood, adipocytes and saline. In this case, the first filter rotates and structurally divides the capacity into two chambers, for the corresponding fractions, and a second filter concentrates the cell fraction before being fed to the mixer. The conductive line allows you to extract the final fraction without violating the tightness of the system and direct it to a centrifuge, then extract the obtained fraction aseptically. The device allows you to enter activators and additives. The system also has a built-in temperature controller. The main disadvantage of this device is that at the first stage of processing, the lipoaspirate is filtered without providing destruction of the stroma of the tissue, which fixes the cells of the stromal-vascular fraction and does not allow them to pass through the filter, thereby reducing the concentration of cells in the permeate. Also, the shortcomings of the system are long trunk lines and a significant number of junctions, which are an environment for adhesion and loss of target cells.
Из предшествующего уровня техники известно устройство (Device for separating adult stem cell US 20130344589 A1 , HUMAN MED AG (DE), ПК C12M1 /00, опубл, 26.12,2013), представляющее собой систему, включающую контейнер для жировой ткани, снабженный устройством для подачи жидкости с целью отмывки биоматериала, смеси необходимых реагентов, попеременно с извлечением фракции содержащей стволовые клетки, клапаном для выравнивания давления, поршнем, вибратором, полупроницаемой мембраной имеющей электростатический заряд и клапаном, которые делят контейнер на 2 части, контроллером температуры. Одна из камер снабжена устройством для перемешивания исходного биоматериала с добавками, совершающего вращательные и/или маятниковые движения. Основным недостатком данного устройства является метод создания давления за счет поршня, что приводит высокой механической нагрузке на целевые клетки из-за продавливания через фильтр и краевого разрушения клеток в месте примыкания поршня к стенке цилиндра. A device is known from the prior art (Device for separating adult stem cell US 20130344589 A1, HUMAN MED AG (DE), PC C12M1 / 00, publ. 12/26/2003), which is a system comprising an adipose tissue container equipped with a feeding device liquids for washing biomaterial, a mixture of necessary reagents, alternately with extracting the fraction containing stem cells, a valve for equalizing pressure, a piston, a vibrator, a semi-permeable membrane having an electrostatic charge and a valve that divide the container into 2 parts, with a temperature controller tours. One of the chambers is equipped with a device for mixing the initial biomaterial with additives, performing rotational and / or pendulum movements. The main disadvantage of this device is the method of creating pressure due to the piston, which leads to a high mechanical load on the target cells due to punching through the filter and edge destruction of the cells at the junction of the piston to the cylinder wall.
В качестве прототипа выбрано устройство для выделения стволовых клеток жировой ткани (System and methods for preparation of adipose-derived stem cells, US 20130012921 A1 , PUSTILNIK FELIX, ПК A61 M37/00, опубл. 10.01 .2013), представляющее собой конусовидную емкость, герметично закрытую крышкой, снабженную разъемами для введения и выведения биоматериала и жидкостей для его обработки, а также вспомогательную пробирку для концентрации,
полученной стромально-васкулярной фракции. Недостатками данной системы являются наличие двух пробирок-емкостей, что увеличивает риск контаминации материала, его потери при перенесении между емкостями, влечет дополнительные требования к вспомогательному оборудованию. Кроме этого, в системе отсутствует разделение первичного липоаспирата на фракции, содержащие целевые клетки и побочные продукты обработки, загрязняющие конечный продукт. A device for the selection of adipose stem cells (System and methods for preparation of adipose-derived stem cells, US 20130012921 A1, PUSTILNIK FELIX, PC A61 M37 / 00, publ. 10.01 .2013), which is a cone-shaped container, is hermetically sealed, as a prototype a closed lid equipped with connectors for the introduction and removal of biomaterial and liquids for its processing, as well as an auxiliary tube for concentration, the resulting stromal-vascular fraction. The disadvantages of this system are the presence of two test tubes, which increases the risk of contamination of the material, its loss when transferred between containers, entails additional requirements for auxiliary equipment. In addition, in the system there is no separation of the primary lipoaspirate into fractions containing target cells and processing by-products polluting the final product.
РАСКРЫТИЕ ИЗОБРЕТЕНИЯ SUMMARY OF THE INVENTION
Технической задачей настоящего изобретения является повышение эффективности выделения стромально-васкулярной фракции с сохранением максимальной жизнеспособности клеток и сохранения ее регенераторного потенциала. The technical task of the present invention is to increase the efficiency of isolation of the stromal-vascular fraction while maintaining maximum cell viability and maintaining its regenerative potential.
Общим техническим результатом изобретения является выделение стромально-васкулярной фракции жировой ткани, характеризующейся высокой концентрацией и жизнеспособностью клеток, отсутствием дебриса и остатков стромы и жировой ткани. A general technical result of the invention is the isolation of the stromal-vascular fraction of adipose tissue, characterized by a high concentration and viability of cells, the absence of debris and residues of the stroma and adipose tissue.
Техническая задача достигается за счет использования герметичного устройства, состоящего из двух камер, расположенных одна над другой и разделенных по крайней мере одним сетчатым фильтром, причем обе камеры представлены в одном корпусе, а нижняя камера объемом 3 - 9 мл имеет килевидную форму, расположена под углом 10 - 45 градусов и вытянута по всей длине устройства. The technical problem is achieved through the use of a sealed device consisting of two chambers located one above the other and separated by at least one strainer, both chambers being in one housing, and the lower chamber with a volume of 3 - 9 ml has a keeled shape, is located at an angle 10 - 45 degrees and extended along the entire length of the device.
Предложенное устройство представляет собой замкнутую герметичную систему в виде прозрачной конусовидной емкости с крышкой объемом 300 - 500 мл, с нанесенной внешней градуировкой по объему и системой внутренних пристеночных каналов, причем емкость разделена, по крайней мере, одним сетчатым фильтром, прилегающим заподлицо к границе раздела камер, на две камеры - рабочая камера для обработки ткани с параболическим дном без острых углов в рабочем пространстве и камера для концентрирования клеток, причем камера для концентрирования клеток имеет объем 3 - 9 мл, имеет килевидную форму, расположена под углом 10 - 45 градусов и вытянута по всей длине устройства.
Устройство имеет, по крайней мере, 6 изолированных каналов, расположенных на боковых стенках емкости с внутренней стороны, причем вход каждого канала имеет крепление для штуцеров-переходников типа «луер-лок». The proposed device is a closed sealed system in the form of a transparent cone-shaped container with a lid with a volume of 300-500 ml, with an external graduation in volume and a system of internal parietal canals, the container being separated by at least one strainer adjacent flush to the interface of the chambers , into two chambers - a working chamber for processing tissue with a parabolic bottom without acute angles in the working space and a chamber for concentrating cells, moreover, a chamber for concentrating cells has t the volume of 3 - 9 ml, has a keeled shape, is located at an angle of 10 - 45 degrees and stretched along the entire length of the device. The device has at least 6 isolated channels located on the side walls of the tank on the inside, and the input of each channel has a mount for Luer-Lock type adapters.
Устройство имеет канал для введения биологического материала, заканчивающийся в рабочей камере для обработки ткани. The device has a channel for introducing biological material, ending in a working chamber for processing tissue.
Устройство имеет канал для введения реагентов и промывочных буферов, заканчивающийся в рабочей камере для обработки ткани. The device has a channel for introducing reagents and wash buffers, ending in a working chamber for processing tissue.
Устройство имеет по крайней мере 3 канала для отбора супернатанта, причем один заканчивается в рабочей камере у места крепления сетчатого фильтра, а два других заканчивается в области 1 /3 - 1 /2 длинны внутренней камеры. The device has at least 3 channels for sampling the supernatant, with one ending in the working chamber at the attachment point of the strainer, and the other two ending in 1/3 - 1/2 of the length of the inner chamber.
Устройство имеет канал для отбора конечного продукта - стромально- васкулярной фракции жировой ткани, заканчивающийся у основания емкости в камере для концентрирования клеток, причем выход канала расположен в остром конце киля камеры для концентирования клеток. The device has a channel for selecting the final product — the stromal-vascular fraction of adipose tissue, ending at the base of the container in the cell concentration chamber, the channel exit being located at the sharp end of the keel of the cell concentration chamber.
Устройство может иметь дополнительные каналы для внесения или отбора компонентов, заканчивающихся в рабочей камере для обработки ткани и/или в камере для концентрирования клеток. The device may have additional channels for introducing or selecting components ending in a working chamber for processing tissue and / or in a chamber for concentrating cells.
Устройство имеет бактериологический фильтр для выравнивания давления внутри системы, причем бактериологический фильтр расположен на крышке устройства. The device has a bacteriological filter for balancing the pressure inside the system, and the bacteriological filter is located on the cover of the device.
Таким образом, биологическая ткань, помещенная в устройство, отмывается от остатков циркулирующей крови; подвергается ферментативной обработке, за счет чего происходит лизис стромальной ткани и выход стромально- васкулярной фракции в среду, разделение клеточного компонента и остатков стромальной и жировой ткани центрифугированием через микрофильтр с последующей отмывкой клеточной фракции от остаточных ферментов и ее концентрирование. За счет параболического дна без острых углов у камеры для обработки ткани и килевидной формы камеры для концентрирования клеток повышается выход клеток. За счет расположения камеры для концентрирования клеток под углом и по всей длине устройства минимизируется риск появления «мертвых» зон и повышается выход клеток. Наличие каналов ввода - вывода с выходами на разном уровне внутри стенки системы позволяет минимизировать
общую площадь внутренней поверхности системы, что снижает риск адгезии клеток на поверхности трубок; позволяет контролируемо пристеночно вносить биологический материал и реактивы, что снижает физическое воздействие на биологический материал и повышает степень выживаемости клеток; позволяет полностью отбирать супернатант с остатками стромы жировой ткани, не затрагивая осадок; позволяет полностью отбирать осадок в виде стромально- васкулярной фракции жировой ткани. Thus, the biological tissue placed in the device is washed from the remnants of the circulating blood; it is subjected to enzymatic treatment, due to which the stromal tissue is lysed and the stromal-vascular fraction enters the medium, the cell component and stromal and adipose tissue residues are separated by centrifugation through a microfilter, followed by washing of the cell fraction from residual enzymes and its concentration. Due to the parabolic bottom without sharp corners, the cell processing chamber and the keeled shape of the cell concentration chamber increase cell yield. Due to the location of the chamber for concentrating cells at an angle and along the entire length of the device, the risk of the appearance of “dead” zones is minimized and the yield of cells is increased. The presence of input / output channels with outputs at different levels inside the system wall allows minimizing the total area of the internal surface of the system, which reduces the risk of cell adhesion on the surface of the tubes; It allows the controlled wall-wise introduction of biological material and reagents, which reduces the physical effect on the biological material and increases the degree of cell survival; allows you to fully select the supernatant with the remains of the stroma of adipose tissue, without affecting the sediment; It allows you to completely select the sediment in the form of a stromal-vascular fraction of adipose tissue.
КРАТКОЕ ОПИСАНИЕ ЧЕРТЕЖЕЙ BRIEF DESCRIPTION OF THE DRAWINGS
На Фиг. 1 - изображен трехмерный общий вид устройства для фракционирования жировой ткани и выделения стромально-васкулярной фракции In FIG. 1 - shows a three-dimensional General view of a device for fractionation of adipose tissue and allocation of stromal-vascular fraction
На Фиг. 2 - изображено устройство для фракционирования жировой ткани и выделения стромально-васкулярной фракции в разрезе ОСУЩЕСТВЛЕНИЕ ИЗОБРЕТЕНИЯ In FIG. 2 shows a device for fractioning adipose tissue and isolating a stromal-vascular fraction in a section.
Сущность изобретения поясняется чертежами, где на Фиг.1 и Фиг.2 изображено устройство для фракционирования жировой ткани и выделения стромально-васкулярной фракции, представляющее собой герметичную емкость (А) с крышкой (Б), разделенную сетчатым микрофильтром (В) на две камеры - камеру для обработки ткани (Г) и камеру для концентрирования клеток (Д). Устройство имеет систему внутренних пристеночных каналов: для внесения липоаспирата (1 ), для внесения реактивов и буферов (2), для отбора супернатанта и излишек жидкости (3-5), для отбора стромально-васкуклярной фракции (6). The invention is illustrated by drawings, where figure 1 and figure 2 shows a device for fractionation of adipose tissue and isolation of the stromal-vascular fraction, which is a sealed container (A) with a lid (B), divided by a mesh microfilter (C) into two chambers - a tissue processing chamber (D) and a cell concentration chamber (D). The device has a system of internal parietal channels: for the introduction of lipoaspirate (1), for the introduction of reagents and buffers (2), for the selection of supernatant and excess fluid (3-5), for the selection of the stromal-vascular layer (6).
ПРИМЕР ОСУЩЕСТВЛЕНИЯ СПОСОБА EXAMPLE OF IMPLEMENTATION OF THE METHOD
Приведенный пример не предназначен для ограничения изобретения, а предложен исключительно в качестве иллюстрации. The above example is not intended to limit the invention, but is offered solely as an illustration.
В общем виде работа устройства представляет собой процесс постадийной отмывки и обработки жировой ткани протеолитическими ферментами с целью выделения стромально-васкулярной фракции, где жировую ткань - липоаспират вносят в устройство - герметичную емкость (А), а именно в камеру для обработки ткани (Г) через канал (1 ). Биологическая ткань отмывается от остатков крови в
буферном растворе, внесенном через канал (2). Излишки жидкости удаляются из системы через канал (3-5). Биологическая ткань подвергается ферментативной обработке смесью протеолитических ферментов, внесенных через канал (2). Устройство подвергается центрифугированию при котором стромально- васкулярная фракция проходит через микрофильтр (В) в камеру для концентрирования клеток (Д). Излишки жидкости удаляются через канал (3-5), осадок отмывается от остатков ферментов буферным раствором. Конечная стромально-васкулярная фракция отбирается через канал (6). In general, the operation of the device is a process of stepwise washing and treating adipose tissue with proteolytic enzymes in order to isolate the stromal-vascular fraction, where adipose tissue - lipoaspirate is introduced into the device - a sealed container (A), namely, into the tissue processing chamber (D) through channel (1). Biological tissue is washed from the remnants of blood in buffer solution introduced through the channel (2). Excess fluid is removed from the system through a channel (3-5). Biological tissue is subjected to enzymatic treatment with a mixture of proteolytic enzymes introduced through the channel (2). The device is subjected to centrifugation in which the stromal-vascular fraction passes through a microfilter (B) into the cell concentration chamber (D). Excess liquid is removed through the channel (3-5), the precipitate is washed from the residual enzymes with a buffer solution. The final stromal-vascular fraction is taken through the channel (6).
Стромально-васкулярную фракцию выделяли из трех образцов липоаспирата, полученного из взрослых здоровых доноров, подписавших добровольное информированное согласие. Забор жира проводили по стандартной методике шприцевой туминесцентной липосакции под местной инфильтрационной анастезией в области передней брюшной стенки. The stromal-vascular fraction was isolated from three lipoaspirate samples obtained from healthy adult donors who signed a voluntary informed consent. Fat sampling was carried out according to the standard technique of syringe tuminescent liposuction under local infiltration anesthesia in the region of the anterior abdominal wall.
Перед ферментативной обработкой липоаспират промывали раствором Хартмана, доводя его объем до 400 мл в колбе. Через 5 минут после добавления раствора Хартмана, всю жидкую часть удаляли шприцем через порт для сбора СВФ. Оставшийся объем жировой ткани измеряли с использованием мерной шкалы на колбе. Ферментативное расщепление проводили добавлением эквивалентного объема раствора коллагеназы NB-6 (GMP Grade, SERVA Electrophoresis GmbH, 0,3 PZ/ml), при 37°C в течение 30 минут при постоянном помешивании. После этого колбу центрифугировали ЗООд, 10 мин, 25°С. После центрифугирования всю жидкость и расщепленный жир (масло) с элементами стромы удаляли через соответствующие порты. Затем процедуру отмывки повторяли еще раз, добавляя 300 мл раствора Хартмана. После последнего центрифугирования весь объем жидкости в верхней камере колбы удаляли шприцем через порты для промывки. Стромально-васкулярную фракцию отбирали шприцем из нижней камеры через порт для сбора СВФ. Объем конечного продукта составлял 10 мл. Before enzymatic treatment, the lipoaspirate was washed with a Hartman solution, bringing its volume to 400 ml in a flask. 5 minutes after adding the Hartman solution, all the liquid part was removed by syringe through the port for collecting SVF. The remaining volume of adipose tissue was measured using a measuring scale on the flask. Enzymatic digestion was performed by adding an equivalent volume of NB-6 collagenase solution (GMP Grade, SERVA Electrophoresis GmbH, 0.3 PZ / ml), at 37 ° C for 30 minutes with constant stirring. After that, the flask was centrifuged with ZOOd, 10 min, 25 ° С. After centrifugation, all liquid and split fat (oil) with stromal elements were removed through the appropriate ports. Then the washing procedure was repeated once more by adding 300 ml of Hartman's solution. After the last centrifugation, the entire volume of liquid in the upper chamber of the flask was removed with a syringe through the washing ports. The stromal-vascular fraction was taken with a syringe from the lower chamber through the port for collecting SVF. The volume of the final product was 10 ml.
Аликвоту СВФ объемом 1 мл использовали для подсчета количества ядросодержащих клеток с помощью гемацитометра. A 1 ml aliquot of SVF was used to count the number of nucleated cells using a hemacytometer.
Для оценки жизнеспособности СВФ использовали окраску клеток трипановым синим.
Для оценки гетерогенности клеточной популяции СВФ использовали моноклональные антитела CD45-PerCp-Cy5.5, CD34-PE-Cy-7, CD146-FITC, CD31 - АРС-Су7, CD90-PE, CD105-APC, CD73-PE, CD133/1 -PE, CD309-APC, CD4-PE, CD14-PerCp-Cy5.5, CD3-APC, CD235-FITC, CD1 17-АРС, NG2-PE (BD Biosciense, США). Окрашивание производили по методике производителя. Анализ полученных популяций проводили с помощью цитофлуориметра FACS Cantoll и програмного обеспечения BD FACS Diva™ Software v.6.1 .3 (BD Biosciense, США). To assess the viability of SVF, trypan blue staining was used. Monoclonal antibodies CD45-PerCp-Cy5.5, CD34-PE-Cy-7, CD146-FITC, CD31 — APC-Su7, CD90-PE, CD105-APC, CD73-PE, CD133 / 1 were used to assess the heterogeneity of the SVF cell population -PE, CD309-APC, CD4-PE, CD14-PerCp-Cy5.5, CD3-APC, CD235-FITC, CD1 17-APC, NG2-PE (BD Biosciense, USA). Staining was performed according to the method of the manufacturer. The analysis of the obtained populations was carried out using a FACS Cantoll cytofluorimeter and BD FACS Diva ™ Software v.6.1.3 software (BD Biosciense, USA).
Таблица 1 . Общая характеристика липоаспирата и стромально-васкулярной фракции, полученной с использованием предложенного устройства Table 1 . General characteristics of lipoaspirate and stromal-vascular fraction obtained using the proposed device
Таблица 2.Table 2.
Фенотипическая характеристика стромально-васкулярной фракции, полученной с использованием предложенного устройства Phenotypic characteristics of the stromal-vascular fraction obtained using the proposed device
жировой ткани (включая adipose tissue (including
супра-адвентициальные CD34+dim CD 1 46-supra adventitious CD34 + dim CD 1 46 -
4.2 6.7 5.9 клетки) (Adipose Derived CD31 CD45" 4.2 6.7 5.9 cells) (Adipose Derived CD31 CD45 "
Stromal Cells (including Stromal cells (including
supraadventitial cells) ) supraadventitial cells))
Гладко-мышечные клетки CD34+d,m CD45- сосудов (Vascular Smooth CD31 " CD146+ 28.7 22.3 26.7 Muscle Cells) CD105+ CD90+ Smooth muscle cells CD34 + d, m CD45 - vessels (Vascular Smooth CD31 " CD146 + 28.7 22.3 26.7 Muscle Cells) CD105 + CD90 +
Неидентифицированные CD34 CD146 CD31 " Unidentified CD34 CD146 CD31 "
18.1 19.0 17.0 клетки (Unidentified Cells) CD45- 18.1 19.0 17.0 cells (Unidentified Cells) CD45-
Общее количество (%) 100 100 100 100 Total amount (%) 100 100 100 100
Хотя настоящее изобретение было подробно описано на примерах вариантов, которые представляются предпочтительными, необходимо помнить, что эти примеры осуществления изобретения приведены только в целях иллюстрации изобретения. Данное описание не должно рассматриваться как ограничивающее объем изобретения, поскольку в этапы описанных устройств специалистами в области медицинской биотехнологии и клеточной технологии и др. могут быть внесены изменения, направленные на то, чтобы адаптировать их к конкретным устройствам или ситуациям, и не выходящие за рамки прилагаемой формулы изобретения. Специалисту в данной области понятно, что в пределах сферы действия изобретения, которая определяется пунктами формулы изобретения, возможны различные варианты и модификации, включая эквивалентные решения. ССЫЛКИ Although the present invention has been described in detail with examples of options that appear to be preferred, it must be remembered that these examples of the invention are provided only for illustrative purposes. This description should not be construed as limiting the scope of the invention, since the steps of the devices described by specialists in the field of medical biotechnology and cell technology, etc., may be amended to adapt them to specific devices or situations and not beyond the scope of the attached claims Specialist in this field it is clear that within the scope of the invention, which is defined by the claims, various options and modifications are possible, including equivalent solutions. LINKS
1 . System for processing lipoaspirate cells ЕР 1921 133 A2 one . System for processing lipoaspirate cells EP 1921 133 A2
2. Method and Apparatus for Separating a Material US 201 10251041 A1 2. Method and Apparatus for Separating a Material US 201 10251041 A1
3. Regenerative cell extraction device WO 2013183797 A1 3. Regenerative cell extraction device WO 2013183797 A1
4. Device for separating adult stem cell US 20130344589 A1 4. Device for separating adult stem cell US 20130344589 A1
5. System and methods for preparation of adipose-derived stem cells, US 20130012921 A1 , WO 201401 1213 A1
5. System and methods for preparation of adipose-derived stem cells, US 20130012921 A1, WO 201401 1213 A1
Claims
ФОРМУЛА ИЗОБРЕТЕНИЯ CLAIM
Устройство для фракционирования жировой ткани и выделения из нее стромально-васкулярной фракции, состоящее из прозрачной конусовидной емкости объемом 300 - 500 мл с крышкой, с нанесенной внешней градуировкой по объему и системой внутренних пристеночных каналов, причем емкость разделена на две камеры - рабочая камера для обработки ткани и камера для концентрирования клеток, отличающееся тем, что камеры разделены по крайней мере одним сетчатым фильтром, прилегающим заподлицо к границе раздела камер, причем камера для обработки ткани имеет параболическое дно без острых углов, а камера для концентрирования клеток объемом 3 - 9 мл имеет килевидную форму, расположена под углом 10 - 45 градусов и вытянута по всей длине устройства.
A device for the fractionation of adipose tissue and the isolation of the stromal-vascular fraction from it, consisting of a transparent cone-shaped container with a volume of 300-500 ml with a lid, with an external graduation in volume and a system of internal parietal channels, with the container divided into two chambers - a working chamber for processing tissue and a chamber for concentrating cells, characterized in that the chambers are separated by at least one strainer adjacent flush to the interface of the chambers, and the chamber for processing tissue has a a rabolic bottom without sharp corners, and a chamber for concentrating cells with a volume of 3 - 9 ml has a keeled shape, is located at an angle of 10 - 45 degrees and is elongated along the entire length of the device.
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| CN113462530A (en) * | 2020-03-31 | 2021-10-01 | 广州盛嘉生物科技有限公司 | Swing type biological tissue treatment device |
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| US20130012921A1 (en) * | 2011-07-08 | 2013-01-10 | Felix Pustilnik | System and methods for preparation of adipose-derived stem cells |
| US20130344589A1 (en) * | 2012-06-22 | 2013-12-26 | Human Med Ag | Device for separating adult stem cell |
| US20160208211A1 (en) * | 2013-09-05 | 2016-07-21 | The Gid Group, Inc. | Tissue processing apparatus and method for processing adipose tissue |
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| RU2252252C1 (en) * | 2004-04-09 | 2005-05-20 | Тепляшин Александр Сергеевич | Method for isolation of mesenchymal stem cells |
| US20130012921A1 (en) * | 2011-07-08 | 2013-01-10 | Felix Pustilnik | System and methods for preparation of adipose-derived stem cells |
| US20130344589A1 (en) * | 2012-06-22 | 2013-12-26 | Human Med Ag | Device for separating adult stem cell |
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