RU2504334C1 - Method of decellularisation of small-calibre blood vessels - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to regenerative medicine and tissue engineering, and can be applied for obtaining extracellular matrixes of small-caliber blood vessels. For this purpose at the first stage of tissues fragment of blood vessel is washed with distilled water for 1 hour at temperature +4°C. After that, fragment is placed into 0.05% solution of tripsin and 0.02% EDTA for 1 hour at temperature +37°C. At the third stage processing in 0.075% solution of sodium dodecylsulfate is performed for 24 hours at temperature 26°C. After that, fragment is placed into 0.25% solution of Triton X-100 for 24 hours at temperature 26°C. At the fourth stage said fragment is processed with solution which contains 20 mcg/ml of RNA-ase and 200 mcg/ml of DNA-ase I, for 6 hours at temperature +37°C. After each stage of processing, fragment of blood vessel is three times washed in phosphate-salt buffer solution, for 10 minutes each time. The entire process of processing is carried out with constant mixing of solutions and simultaneous vibration, created by vibromotor, located on the external wall of the reservoir.

EFFECT: method makes it possible to increase quality of decellularisation of small-calibre blood vessels, preserve their integrity and ultrastructure for further immobilisation of recipient's cells.

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Description

The invention relates to the field of biology and medicine, namely to the field of regenerative medicine and tissue engineering, and can be used to obtain extracellular matrixes of organs and tissues used in reconstructive surgery to replace or replace the functions of the affected organs and tissues.

Extracellular matrix (ECM), which is used in tissue engineering of organs and tissues, can be obtained by the method of decellularization. Known methods of decellularization involve sequential multi-stage treatment with solutions of detergents and / or enzymes (Amiel GE, Komura M, Shpira O, Yoo JJ, Yazdani S. Berry J, et al. Engineering of blood vessels from acellular collagen matrices coated with human endothelial cells. Tissue Eng 2006, 12: 2355; C. Williams, J. Liao, EMJoyce, B. Wang, JB Leach, MS Sacks and JYWong. Altered structural and mechanical properties in decellularized rabbit carotid arteries, Acta Biomater. 5, 993-1005 (2009); patent US 8221777 "Structurally modified acellular tissue engineering scaffolds and methods of production same"; patent RU No. 2407459, 2009) and differ in the duration of exposure and concentration of reagents, which use such solutions, like Triton X-100, trypsin with ethylenediaminetetraacetic acid (EDTA), phosphate-buffered saline, nucleases. As a rule, the tissue being treated is placed in a container with a reagent during decellularization, while the reagent is constantly mixed for a sufficiently long time.

However, the known methods of decellularization have disadvantages in that the cells and their residues (cell debris) are not completely removed from the treated tissue, and serious damage to the ECM is not ruled out.

Of the known analogues, the method Amiel GE, Komura M, Shpira O, Yoo JJ, Yazdani S. Berry J, et al. Was selected as a prototype of the claimed method of decellularization. Engineering of blood vessels from acellular collagen matrices coated with human endothelial cells. Tissue Eng 2006, 12: 2355, which consists in the fact that a fragment of a blood vessel is gradually phased in a container with:

- distilled water for 24 hours at a temperature of + 4 ° C,

- 0.05% trypsin-EDTA solution for 1 hour,

- DMEM nutrient medium with 5% serum for 24 hours,

- 1% solution of Triton X-100 with 0.1% ammonium hydrochloride (ammonia, ammonia) for 3 days at a temperature of + 4 ° C, while at this stage the processing process is carried out with constant stirring of the solution using a shaker,

- distilled water for 24 hours at a temperature of + 4 ° C,

- phosphate-buffered saline for 24 hours at a temperature of + 4 ° C.

The known method has the same disadvantages as its analogues, namely, it does not completely remove the cells and their residues from the processed material, and does not exclude damage to the cell matrix, since it is not designed to process small blood vessels.

The objective of the present invention is to improve the quality of decellularization of small blood vessels, preserving their integrity and ultrastructure for subsequent immobilization of the recipient cells.

The achievement of this task is achieved by the fact that in the method of small cell blood vessel decellularization, namely, distilled water, a 0.05% trypsin-EDTA solution, Triton X-100 solution, and Triton X-100 are used to remove cellular elements from the processed fragment of the blood vessel. phosphate-saline buffer solution, a fragment of a blood vessel is sequentially phased placed in a container with:

distilled water for 1 hour at a temperature of + 4 ° C,

0.05% trypsin and 0.02% EDTA for 1 hour at a temperature of + 37 ° C,

0.075% sodium dodecyl sulfate solution for 24 hours at a temperature of 26 ° C,

0.25% solution of Triton X-100 for 24 hours at a temperature of 26 ° C,

a solution containing Rnase A nuclease 20 μg / ml and DNase I 200 μg / ml,

after each stage of processing, a fragment of a blood vessel is washed three times in phosphate-buffered saline for 10 minutes, and the entire processing process is carried out with constant stirring of the solutions and the simultaneous vibration created by the vibromotor located on the outer wall of the tank.

The inventive method of decellularization of blood vessels of small caliber differs from analogues and prototype in combination, sequence and duration of use of decellularizing agents, their lower concentration and the presence of additional physical effects - vibration created by a vibration motor mounted on the outer wall of the tank, which allows more efficient removal of cells from the processed fragment blood vessel with less damage to the ECM.

The proposed method for the decellularization of blood vessels of small caliber (3-4 mm), which used human umbilical arteries in the experiment, involves several stages with the change of decellularizing solutions (DR), which use detergents and enzymes. All stages are carried out with a constant rotational movement of the DR in the working capacity, for which the capacity with the DR is installed on the orbital shaker at a speed of 30 rpm and a movement amplitude of 10 mm. In addition, for the deepest penetration of DR into the thickness of the vascular wall and more complete removal of cellular elements, vibration is simultaneously used. To create vibration, a small motor with an eccentric (vibromotor) is installed on the outside of the working tank with DR.

As a result of studies of small-caliber blood vessels decellularized by the described method, it can be concluded that the invention provides the achievement of the task, which is to improve the quality of small-caliber blood vessels decellularization, preserving their integrity and ultrastructure for subsequent immobilization of recipient cells.

The invention is new and industrially applicable, as it can be used to obtain extracellular matrixes of organs and tissues for subsequent use in reconstructive surgery to replace or replace prosthetic functions of affected organs and tissues.

The invention is illustrated by drawings, where:

figure 1 presents a cross section of the native artery of the human umbilical cord, the color of hematoxylin-eosin, magnification × 100, where 1 - cell elements, 2 - connective tissue;

figure 2 is a cross section of a decellularized artery of the human umbilical cord, stained with hematoxylin-eosin, magnification × 100, where: 2 - connective tissue (extracellular matrix). Cellular elements are not visualized;

figure 3 is a transverse section of the native artery of the human umbilical cord, the color according to Van Gieson, magnification × 200, where 1 is the cellular elements; 2 - connective tissue;

figure 4 is a cross-section of a decellularized artery of the human umbilical cord, Van Gieson stain, magnification × 200, where 2 are collagen fibers (connective tissue), 3 are gaps;

figure 5 is a transverse section of the native artery of the human umbilical cord, immunohistochemical staining for smooth muscle actin antigen (a-SMA), magnification × 100; where: 1 - smooth muscle cells;

figure 6 is a transverse section of a decellularized artery of a human umbilical cord, immunohistochemical staining for α-smooth muscle actin antigen, magnification × 200, where 2 is connective tissue (extracellular matrix). Smooth muscle cells and their debris are not visualized.

The method is as follows.

At the first stage, a fragment of a blood vessel, which is used as a human umbilical artery, is washed from the blood in disciled water for 1 hour at a temperature of + 4 ° С. Since water is a hypotonic solution, osmotic shock and cell lysis occur. Then the fragment of the treated artery is washed three times in a buffer solution for 10 minutes. After each step, 0.1 M phosphate-saline buffer solution with the addition of 0.02% EDTA was used as a washing solution.

At the second stage, a solution of trypsin and EDTA is used for 1 hour, at a temperature of + 37 ° C. The solution contains 0.05% trypsin, 0.02% EDTA in 0.1 M phosphate-buffered saline without Ca2 + and Mg2 + ions. In cell biology, a mixture of trypsin and EDTA is widely used to separate cells from the surface of the substrate, both at a concentration of 0.05% and 0.25%. In the claimed method uses a lower concentration of trypsin to prevent proteolysis of matrix proteins. Trypsin leads to the separation of cells from the ECM and the "loosening" of the matrix microstructure for deeper penetration of detergents and to improve the removal of cells from the thickness of the vascular wall in further stages of decellularization. The functions of EDTA are mediated by the binding of Ca2 + and Mg2 + ions, which, firstly, can inhibit the trypsin effect, secondly, are necessary for cell adhesion to the extracellular matrix, and thirdly, EDTA non-selectively inhibits matrix metalloproteinases, which prevents ECM self-destruction.

In the third stage, after washing the vessel fragment three times in phosphate-buffered saline for 10 minutes, the solution is treated with sodium dodecyl sulfate for 24 hours at a temperature of + 26 ° C. DR at this stage contains 0.075% SDS and 0.02% EDTA in ODM phosphate-buffered saline. Sodium lauryl sulfate (English sodium lauryl sulfate, SLS) or sodium dodecyl sulfate (English sodium dodecyl sulfate, SDS) is a sodium salt of lauryl sulfonic acid, an anionic surfactant. It is used for solubilization of cell membrane proteins and cell lysis, followed by leaching of cell debris from the matrix. For the best effect of decellularization, it is possible to change the solution after 1-2 hours.

In the fourth stage, after washing three times in phosphate-buffered saline for 10 minutes, the treatment is carried out with Triton X-100 solution for 24 hours at a temperature of + 26 ° C. DR at this stage contains 0.25% Triton X-100 and 0.02% EDTA in 0.1 M phosphate-buffered saline. Triton X-100 is a non-ionic surfactant. It contains hydrophobic (4-tert-octylphenol) and hydrophilic fragments (from residues of ethylene oxide, in the amount of 9-10). Triton X-100 is a weaker detergent than SDS. It is also used for solubilization of cell membrane proteins and cell lysis, followed by leaching of cell debris from the matrix. Also to remove residual SDS, which is sufficiently cytotoxic for subsequent recellularization.

The fifth stage, after the next three times washing in phosphate-buffered saline for 10 minutes, consists in the destruction of nucleic acids, which are responsible for the immune response in the recipient. Nucleases are used for this, namely DNase I (200 μg / ml) and RNase A (20 μg / ml) for 6 hours at a temperature of + 37 ° C.

The final stage consists in washing the area of the artery to be treated three times in phosphate-buffered saline to finally remove the detergents and inactivate the enzymes.

Example 1

To obtain the extracellular matrix of a small blood vessel, a human umbilical artery was used. The material was obtained directly in the maternity ward of the Federal Specialized Perinatal Center of FSBE named after VA Almazova ”and at a temperature of + 4 ° C was transported to the laboratory. Delivery time was no more than 2 hours. Under sterile conditions, an artery fragment of about 5 cm long was extracted using tweezers and scissors from the umbilical cord. Decellularization was carried out in several stages with constant stirring of the decellularizing solution (DR), for which a container with DR was installed on an orbital shaker at a speed of 30 revolutions per minute and the amplitude of movements 10 mm (orbital shaker GFL 3005, GFL, Germany), and additionally created vibration, using a small motor with an eccentric mounted on the outside of the working tank with DR (QX-6A-1 vibromotor, QX Motor, China) . The first stage was the washing of the blood vessel section from the blood in distilled water for 1 hour at a temperature of + 4 ° С. Then, the blood vessel section was sequentially placed in a container with a 0.05% trypsin solution (Samson-Med, Russia) in phosphate-buffered saline with ethylenediaminetetraacetic acid (FSB-DTA) for 1 hour at a temperature of + 37 ° C, 0.075% sodium dodecyl sulfate solution (Amresco, USA) for 24 hours at a temperature of + 26 ° C; 0.25% solution of Triton X-100 (Amresco, USA.) For 24 hours at a temperature of + 26 ° C; with nucleases (RNase A 20 μg / ml. and DNase I 200 μg / ml, Rosmedbio, Russia). After each stage, the artery section was washed three times in FSB-EDTA for 10 minutes. After the final three-time washing, sections of the arteries were examined.

Example 2

To obtain the extracellular matrix of a small blood vessel, a frozen human umbilical artery was used. The material was obtained directly in the maternity ward of the Federal Specialized Perinatal Center of FSBE named after V.A. Almazova ”and at a temperature of + 4 ° C was transported to the laboratory. Delivery time was no more than 2 hours. Under sterile conditions, using both tweezers and scissors from the umbilical cord, both arteries were separated, which were divided into sections about 5-6 cm long. The sections of the arteries were then frozen and stored at a temperature of -80 ° C until their subsequent use. The duration of storage in the frozen state can be up to 6 months. To carry out decellularization, the umbilical artery fragment was thawed in a water bath at a temperature of + 37 ° С, the treatment by the proposed method was carried out and used in further studies.

Quality control of decellularization by the present method was carried out using histological and immunohistochemical analysis, measurement of residual DNA and RNA.

Figure 1 presents the morphological picture of the wall of the intact artery of the human umbilical cord stained with hetoxylin-eosin, where you can see the characteristic structure of the vascular wall rich in cellular elements. Figure 2 presents the artery of the human umbilical cord after decellularization according to the claimed method. As you can see, the cellular elements are completely absent, and the extracellular matrix has an intact structure. The extracellular matrix is seen in more detail in FIGS. 3 and 4, where histological preparations are stained according to Van Gieson, used to stain connective tissue, especially collagen fibers, of which the extracellular matrix of the artery mainly consists. To assess the completeness of removal of cell debris from the thickness of the vascular wall, the preparations were stained for smooth muscle actin antigen - Figures 5 and 6. Figure 5 shows a thick array of smooth muscle cells in the middle layer of the artery (muscle type artery), after decellularization by the claimed method, smooth muscle actin antigen it is not visually detected, which indirectly confirms not only the lysis of smooth muscle cells, but also the removal of cell debris from the entire thickness of the vascular wall.

To compare the amount of nucleic acids in native and decellularized arteries, DNA and RNA were extracted from fragments of blood vessels. Quantitative analysis was performed on a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, USA). In the native artery, the DNA level was 81.9ng / U1, RNA was 98.4 ng / U1, and neither DNA nor RNA were detected in the decellularized artery.

The effectiveness of the proposed method is tested in a series of 20 consecutive experiments, while achieving reproducible results. Thus obtained decellularized blood vessels can be used in further stages of tissue engineering to create autologous vascular substitutes in reconstructive cardiovascular surgery.

Claims (1)

The method of small-caliber blood vessel decellularization, which consists in the fact that distilled water, a 0.05% trypsin-EDTA solution, Triton X-100 solution and phosphate-saline buffer solution are used to remove cellular elements from the blood vessel, characterized in that a fragment of a blood vessel is sequentially phased placed in a container with:
distilled water for 1 hour at a temperature of + 4 ° C,
0.05% trypsin and 0.02% EDTA for 1 hour at a temperature of + 37 ° C,
0.075% sodium dodecyl sulfate solution for 24 hours at a temperature of 26 ° C,
0.25% solution of Triton X-100 for 24 hours at a temperature of 26 ° C,
a solution containing Rnase A nuclease 20 μg / ml and DNase I 200 μg / ml for 6 hours at a temperature of + 37 ° C,
after each stage of processing, a fragment of a blood vessel is washed three times in phosphate-buffered saline for 10 minutes, and the entire processing process is carried out with constant stirring of the solutions and the simultaneous vibration created by the vibromotor located on the outer wall of the tank.

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