RU173796U1 - DEVICE FOR FACTION OF FAT TISSUE AND ISOLATION FROM HER STROMAL-VASCULAR FACTION FOR APPLICATION IN REGENERATIVE MEDICINE - Google Patents

Abstract

The proposed device relates to medical biotechnology and cell technology and is intended for fractionation of adipose tissue and isolation of the stromal-vascular fraction from it for regenerative medicine. The technical result of the utility model is the controlled isolation of the stromal-vascular fraction of adipose tissue, characterized by high cell survival, lack of debris, residues of stromal and adipose tissue and circulating blood cells, low concentration and activity of residual enzymes . 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, the lower chamber having a keeled shape, located at an angle and elongated along the entire length of the device.

Description

Technical field

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.

State of the art

Intact adipose tissue is a tissue rich in blood vessels, consisting of mature fat cells (adipocytes), a stromal-vascular cell fraction and supporting stroma.

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.

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.

Since that time, numerous studies have been conducted on the clinical possibilities of using SVFs and ADSCs, which have shown 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.

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.

A known method of isolating stem cells from adipose tissue using the manual method (Zuk PA, Zhu M., Mizuno H. et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng. 2001; 7 (2): 211-28). This method is based on the treatment of lipoaspirate with 0.075% type I collagenase solution at a temperature of 37 ° C for 30 minutes and subsequent centrifugation of the resulting suspension at 800 g. After centrifugation, the cell suspension is divided into two fractions: adipocytes are in the upper supernatant layer, and the stromal-vascular fraction with an admixture of red blood cells, which were removed during incubation in a lysis solution of ammonium chloride, is in the sediment. 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 1921133 A2, CYTORI THERAPEUTICS, INC (US), IPC A61K31 / 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. At the same time, the first filter rotates and structurally divides the container into two chambers, for the corresponding fractions, and the 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 send it to a centrifuge, then aseptically extract the obtained fraction. 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 prior art device is known (Device for separating adult stem cell US 20130344589 A1, HUMAN MED AG (DE), IPC C12M1 / 00, published December 26, 2013), which is a system including an adipose tissue container equipped with a device for feeding liquids for washing biomaterial, a mixture of necessary reagents, alternately with extracting the fraction containing stem cells, a pressure balancing valve, a piston, a vibrator, a semi-permeable membrane that has an electrostatic charge, and a valve that divide the container into 2 parts, a tempo controller ratra. 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 isolation of adipose stem cells (System and methods for preparation of adipose-derived stem cells, US 20130012921 A1, PUSTILNIK FELIX, IPC A61M37 / 00, publ. 10.01.2013), which is a cone-shaped container, hermetically closed, is selected as a prototype a lid equipped with connectors for the introduction and removal of biomaterial and liquids for its processing, as well as an auxiliary tube for the concentration of the obtained 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 during transfer between containers entail 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.

Disclosure of invention

The technical task of this utility model is to increase the efficiency of isolation of the stromal-vascular fraction while maintaining maximum cell viability and maintaining its regenerative potential.

The general technical result of the utility model 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 the same 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 airtight system in the form of a transparent cone-shaped container with a lid of 300–500 ml, with an external graduation in volume and a system of internal wall channels, 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 sharp angles in the working space and a chamber for concentrating cells, and a chamber for concentrating cells has it has 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.

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, one of which ends in the working chamber at the attachment point of the strainer, and the other two end in the region 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.

Thus, the biological tissue placed in the device is washed from the remnants of the circulating blood; 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 tube surface; 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; allows you to fully select the sediment in the form of a stromal-vascular fraction of adipose tissue.

Brief Description of the Drawings

In FIG. 1 shows a three-dimensional general view of a device for fractionating adipose tissue and isolating the stromal-vascular fraction.

In FIG. 2 shows a device for fractioning adipose tissue and isolating a stromal-vascular fraction in a section.

The implementation of the invention

The essence of the utility model is illustrated by the drawings, in which Fig. 1 and Fig. 2 show a device for fractioning adipose tissue and isolating the stromal-vascular fraction, which is a sealed container (A) with a lid (B), divided by a mesh microfilter (C) into two chambers - a chamber for tissue processing (G) and a chamber for concentrating cells (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).

An example of the method

The above example is not intended to limit the invention, but is offered solely as an illustration.

In general terms, 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 (G), through the channel (1). Biological tissue is washed off from the remnants of blood in a buffer solution introduced through a 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).

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.

Before enzymatic treatment, the lipoaspirate was washed with a Hartman solution, bringing its volume to 400 ml in a flask. 5 minutes after the addition of the Hartman solution, all of the liquid part was removed by syringe through the SVF collection port. 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 300g, 10 min, 25 ° C. 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.

A 1 ml aliquot of SVF was used to count the number of nucleated cells using a hemacytometer.

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-Cy7, 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, CD117-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 CantoII cytofluorimeter and BD FACS Diva ™ Software v.6.1.3 software (BD Biosciense, USA).

Table 1.

General characteristics of lipoaspirate and stromal-vascular fraction obtained using the proposed device

Indicator Sample No. 1 Sample No. 2 Sample No. 3 Donor age 48 54 67 Floor F F M The volume of lipoaspirate, ml 98 146 78 The number of nucleated cells, million 77 104 52 Viability,% 92 93 95

Table 2.

Phenotypic characteristics of the stromal-vascular fraction obtained using the proposed device

Although the present utility model has been described in detail with examples of options that appear to be preferred, it must be remembered that these embodiments of the utility model are provided only to illustrate the invention. This description should not be construed as limiting the scope of the utility model, 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 exceed the scope of the attached utility model formulas. Specialist in this field it is clear that within the scope of the utility model, which is determined by the claims, various options and modifications are possible, including equivalent solutions.

References

1. System for processing lipoaspirate cells EP 1921133 A2.

2. Method and Apparatus for Separating a Material US 20110251041 A1.

3. Regenerative cell extraction device WO 2013183797 A1.

4. Device for separating adult stem cell US 20130344589 A1

System and methods for preparation of adipose-derived stem cells, US 20130012921 A1, WO 2014011213 A1.

Claims (1)

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 the arabolic bottom without sharp corners, and the 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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