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WO2018160096A1 - Dispositif pour fractionner le tissu adipeux et en extraire la fraction vasculaire stromale utilisée en médecine régénérative - Google Patents

Dispositif pour fractionner le tissu adipeux et en extraire la fraction vasculaire stromale utilisée en médecine régénérative Download PDF

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Publication number
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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WIPO (PCT)
Prior art keywords
stromal
adipose tissue
vascular fraction
cells
tissue
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PCT/RU2017/050097
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English (en)
Russian (ru)
Inventor
Алексей Владимирович ВЕРЕМЕЕВ
Илья Игоревич ЕРЕМИН
Владимир Георгиевич НЕСТЕРЕНКО
Роман Николаевич БОЛГАРИН
Полина Андреевна КОЛОМЕНСКАЯ
Original Assignee
Общество С Ограниченной Ответственностью "Джоин Тексэлл"(Ооо "Джоин Тексэлл")
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Publication of WO2018160096A1 publication Critical patent/WO2018160096A1/fr

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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
    • 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

Le dispositif de l'invention concerne le domaine des biotechnologies médicales et cellulaires et est destiné à fractionner le tissu adipeux et en extraire la fraction vasculaire stromale utilisée en médecine régénérative. Le résultat technique de l'invention consiste en un dégagement contrôlé de la fraction vasculaire stromale du tissu adipeux caractérisé par un taux de survie élevé des cellules, une absence de débris, de résidus des tissus stromal et adipeux et de cellules du sang en circulation, de faibles concentration et activité des ferments résiduels. Le résultat technique consiste à utiliser un dispositif étanche constitué de deux chambres divisées l'une au-dessus de l'autre et séparées par au moins un filtre en grille, la chambre inférieure possédant une forme de quille étant disposée à un angle et allongée sur toute la longueur du dispositif.
PCT/RU2017/050097 2017-03-02 2017-09-29 Dispositif pour fractionner le tissu adipeux et en extraire la fraction vasculaire stromale utilisée en médecine régénérative WO2018160096A1 (fr)

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RU2017106932 2017-03-02

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113462530A (zh) * 2020-03-31 2021-10-01 广州盛嘉生物科技有限公司 一种摇摆式生物组织处理装置

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2252252C1 (ru) * 2004-04-09 2005-05-20 Тепляшин Александр Сергеевич Способ выделения мезенхимальных стволовых клеток
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

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2252252C1 (ru) * 2004-04-09 2005-05-20 Тепляшин Александр Сергеевич Способ выделения мезенхимальных стволовых клеток
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

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113462530A (zh) * 2020-03-31 2021-10-01 广州盛嘉生物科技有限公司 一种摇摆式生物组织处理装置

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