RU2714327C1 - Method for accelerated decellularization of biological tissue or organ - Google Patents

Abstract

FIELD: medicine; biology.

SUBSTANCE: invention refers to biology and medicine, particularly to regenerative medicine and tissue engineering, and can be used for rapid production of extracellular matrixes of organs and tissues used in reconstructive surgery for replacement and prosthetics of the involved organs and tissues. Method for decellularization of biological tissue or organ involves removing cell elements by washing biological tissue or organ with a solution containing tris(hydroxymethyl)aminomethane, sodium chloride, sodium dodecyl sulphate, sodium deoxycholate, triton X-100, and deionized water with the following content of components, wt%: tris(hydroxymethyl)aminomethane 1.7–4.1, sodium chloride 4.62–11.62, sodium dodecyl sulphate 0.08–0.16, sodium deoxycholate 0.25–0.65, triton X-100 0.65 –1.25, deionized water up to 100.

EFFECT: disclosed is a method for declerularisation of biological tissue.

1 cl, 3 dwg, 1 tbl, 2 ex

Description

Technical field

The invention relates to the field of biology and medicine, in particular, the field of regenerative medicine and tissue engineering, and can be used for express production of extracellular matrixes of organs and tissues used in reconstructive surgery to replace and replace the volume of the affected organs and tissues.

State of the art

The extracellular matrix is used in tissue engineering of organs and tissues as a cell carrier for the formation of an artificial cell niche. The extracellular matrix is the basis of connective tissue and can be obtained by the method of decellularization. The problem of obtaining a decellularized extracellular matrix is associated with the difficulty in selecting suitable methods and exposure times that preserve the mechanical and biological properties of the matrix, and at the same time lead to the degradation of donor cells and residues of chemical detergents used in the process of decellularization. Known methods of decellularization involve sequential multi-stage treatment with solutions of detergents and / or enzymes [Zhou P., et al. (2016). Decellularization and recellularization of rat livers with hepatocytes and endothelial progenitor cells. Artificial organs, 40 (3), E25-E38; Mazza G., et al. (2015). Decellularized human liver as a natural 3D-scaffold for liver bioengineering and transplantation. Scientific reports, 5, 13079; Maghsoudlou P., et al. (2013). A decellularization methodology for the production of a natural acellular intestinal matrix. Journal of Visualized Experiments, (80), e50658] and differ in the duration of exposure and the concentration of reagents, which are used as solutions such as sodium dodecyl sulfate, Triton X-100, sodium deoxycholate, NaCl hypertonic solution with a concentration of more than 0.9%. 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.

A known method of decellularization of the esophagus of experimental animals to create a bioengineered design [Patent for invention RU 2662554], which consists in the decellularization of detergents and enzymes in the following sequence: deionized water for 1.5 hours, then use a 4% solution of sodium deoxycholate in combination with 0.002 M Na -EDTA for 70 minutes, phosphate buffer for 15 minutes, pork pancreatic DNase I 2000 U / 200 ml phosphate buffer with calcium and magnesium for 70 minutes, complete the decellularization by washing with decellularizing solutions and disinfection of the matrix in a 10% solution of chlorhexidine bigluconate in phosphate buffer with a change in solution and perfusion direction every 5 hours

A known method for the decellularization of rat heart [Patent for invention RU 2550286], which consists in perfusion through the aorta with purified water, phosphate-buffered saline, detergents and enzymes: solutions containing 4% sodium deoxycholate solution in combination with 0.002M Na2-EDTA, pork pancreatic DNase-I 2000 IU / 200 ml of phosphate buffer with calcium and magnesium and washing the scaffold with phosphate buffer with the addition of 1% penicillin-streptomycin with a total duration of 28 hours.

Known methods of decellularization have disadvantages in that the tissue to be decellularized is located for a long time in the reaction zone, in which serious structural damage to the extracellular matrix is not excluded, and the cellular elements are not completely removed.

The closest analogue is “The method for producing decellularized matrices of parenchymal organs of laboratory animals” [Patent for invention RU 2653489], which consists in obtaining decellularized matrices of the kidneys of rabbits or rats using the method of detergent-enzymatic decellularization using a high-performance perfusion system for 5 stages, namely : washing organs from residual blood 1X PBS with heparin for 1 hour; perfection of the rabbit kidney parenchyma with a hypotonic buffer of 10 mM HEPES sodium salt, 1 mM Mg2SO4 * 6H2O, 0.4% KCL for 5-6 hours, or rat kidney 1X Tris EDTA in 0.1 M PBS without calcium and magnesium; perfusion of organ parenchyma with a solution containing ionic (0.25% sodium dodecyl sulfate SDS, 0.25% sodium deoxycholate (SDC)) and non-ionic (Triton X-100 0.25% for rabbit kidney, 0.1% SDS, 0.1 % SDC and 0.25% TNX for rat kidney) detergents within 24 hours; sequential perfusion of organs with detergent solutions in increasing concentrations for 16 hours for the rabbit kidney 0.3% SDS, SD and TNX, 0.4%; 0.4% SDS, SD and TNX; for rat kidney - 0.25% SDS, SD and TNX; 0.3 SDS, SD and TNX followed by washing the organs from trace amounts of 1X PBS detergent for 1.5 hours.

The disadvantage of this method is the length of the process of decellularization, due to the insufficiently high concentration of detergents, which can lead to the loss of microarchitectonics extracellular matrix.

Disclosure of Invention

The technical problem to which the present invention is directed is the development of a method for accelerated decellularization of various organs and tissues, while maintaining their integrity and ultrastructure for subsequent immobilization on a decellularized matrix of recipient cells.

The technical result of the invention is to reduce the time of decellularization of organs and tissues while maintaining their integrity and ultrastructure.

The technical result is achieved by developing a method of decellularization of a biological tissue or organ, including the removal of cellular elements by washing the biological tissue or organ with a solution containing tris (hydroxymethyl) aminomethane, sodium chloride, sodium dodecyl sulfate, sodium deoxycholate, Triton X-100, and deionized water at the following content of components, mass. %:

Tris (hydroxymethyl) aminomethane 1.7-4.1 Sodium chloride 4.62-11.62 Sodium dodecyl sulfate 0.08-0.16 Sodium deoxycholate 0.25-0.65 Triton X-100 0.65-1.25 Deionized water up to 100

In this case, the biological tissue or organ is selected from the group consisting of the liver, blood vessel, trachea, lung, heart, intestines, stomach, and bile duct.

The inventive method of decellularization differs from analogues in combination, concentration, sequence and duration of use of decellularizing agents.

The invention is illustrated by illustrations, where in FIG. 1 shows a microscopic photograph of a histological specimen of the decellularized matrix liver of the Wistar rat liver (magnification × 200, size scale 50 μm); in FIG. 2 - tissue-engineering design implanted in the rabbit: (A) formation of a tracheal defect - resection of 5 rings in the tracheal wall of the rabbit; (B) tissue engineering design implanted in a tracheal defect; in FIG. 3 - rabbit tracheal tissue after 6 months. after implantation of a tissue engineering construct, hematoxylin-eosin staining. Overview image of tracheal tissue from two sides relative to the incision.

The implementation of the invention

The proposed method is as follows.

A tissue or organ is prepared for decellularization as follows. Freshly obtained resection material is preliminarily washed from the blood by perfusion with Ringer's solution with 0.2 ± 0.2% heparin or warfarin, or with isotonic saline with 0.2 ± 0.2% heparin or warfarin, or 0.9% sodium chloride solution for injection with 0.2 ± 0.2% heparin or warfarin at a temperature of 35 ± 10 ° С at a speed of 40 ± 20 ml / min for 20 ± 5 min, and then washed with phosphate buffer (PBS) with a pH of 7.2 ± 1.5 or with isotonic saline solution, or 0.9% NaCl solution for injection at a temperature of 35 ± 5 ° C at a rate of 40 ± 20 ml / min for 10 ± 5 minutes

The method of decellularization of a tissue or organ in accordance with the present invention is as follows. The biological tissue or organ is treated with a solution of 1.7-4.1% hydroxymethylaminomethane (Tris), 4.62-11.62% sodium chloride (NaCl), 0.08-0.16% sodium dodecyl sulfate (SDS), 0.25-0.65 % sodium deoxycholate (SDC), 0.65-1.25% Triton X-100 in deionized water for at least 15 minutes Sodium dodecyl sulfate (SDS), sodium deoxycholate (SDC) are anionic surfactants used to solubilize cell membrane proteins and cell lysis, followed by leaching of cellular material from the matrix. Tris (born Tris) - a buffer component that allows you to keep the pH during the procedure at a biological level. Sodium chloride (NaCl) - a salt of hydrochloric acid, serves to maintain ionic strength during the procedure at a given level. Triton X-100 (Eng. Triton X-100) is a nonionic surfactant used for binding to cell membranes and their destruction, as well as lysis of cells, followed by leaching of cellular material from the matrix. This combination of agents allows you to efficiently and quickly remove cellular material, almost without affecting the extracellular matrix due to the short interaction time and constantly maintained pH, allowing the protein to be in a single ionic form throughout the procedure and constantly maintained ionic strength, due to which the binding of detergents and proteins difficult. This method allows you to remove accelerated cellular components from an organ or tissue.

In the practice of the present invention, further manipulations can be used to improve other product qualities besides removing the cellular component, for example, perfusion with phosphate buffer (PBS) with a pH of 7.2 ± 1.5 or isotonic saline, or 0.9% NaCl for injection at a temperature of 35 ± 5 ° С at a speed of 40 ± 20 ml / min for 10 ± 5 min or washing with a PBS solution from an antibiotic-antimycotic at a concentration of 10X (Gibco, Cat No. 15240062, USA) at 4 ° С, flow rate 40 ± 20 ml / min for 15 ± 5 minutes.

Below are examples of the implementation of the proposed method, where the rat liver was used as a decellularized organ.

Example 1. Carrying out the decellularization of organs and tissues.

The animal (Wistar rats) was euthanized with xylazine solution and an autopsy was performed. All solutions used in the work were sterilized by filtration through a 0.22 μm filter. The organ was carefully removed from the abdominal cavity without damaging the capsule. Decellularization was performed using a system composed of a peristaltic pump (MasterFlex L / S, USA), a reservoir with perfusion solution, silicone tubes of 18-26 diameter (MasterFlex, USA) and a threaded adapter to the catheter. A catheter was inserted into the inferior vena cava, then connected to a peristaltic pump and the organ was washed with solutions filled into the reservoir of the system for decellularization, as follows.

1. Perfusion of the liver was performed to wash off residual blood with isotonic saline with 0.2% heparin at a temperature of 35 ° C at a rate of 40 ml / min for 20 minutes

2. Washed from heparin when perfused with phosphate buffer with a pH of 7.2 at a temperature of 35 ° C at a speed of 40 ml / min for 10 minutes

3. Decellularization was carried out with a solution containing 2.9% Tris, 8.12% sodium chloride, 0.12% sodium dodecyl sulfate, 0.45% sodium deoxycholate, 0.95% Triton X-100 at a temperature of 37 ° C and a flow rate of 40 ml / min for 15 minutes.

4. Washing from detergents was carried out using a solution of phosphate buffer at a temperature of 37 ° C, a flow rate of 40 ml / min for 5 minutes.

5. Additionally, washing with a PBS solution with an antibiotic-antimycotic at a concentration of 10X (Gibco, Cat No. 15240062, USA) was carried out at 4 ° C, a flow rate of 40 ml / min for 10 minutes to prevent bacterial contamination.

The quality of the obtained material in the form of a decellularized rat liver was evaluated by DNA counting and microscopy of histological preparations. During histological analysis, sections were stained according to Masson, to visualize collagen fibers. No morphologically preserved cells were observed in the decellularized preparation; the tissue is represented mainly by the fibrous structure formed by the extracellular matrix and the vessels of the liver. The residual dsDNA was 21.9 ± 2.4 × 10 ^-3 ng per 1 μg of the sample, which indicates the qualitative removal of the cellular component from the extracellular matrix.

As a result of the studies of organs decellularized in the described manner, it can be concluded that the invention provides the achievement of the task, expressed in reducing the time of organ decellularization - the total time of decellularization was 80 ± 35 minutes, while the total time of decellularization in the prototype is 47, 5 hours, improving the quality of organ decellularization, maintaining their integrity and ultrastructure for subsequent immobilization of recipient cells.

The effectiveness of the proposed method is tested in a series of 7 consecutive experiments, while achieving reproducible results.

The table below shows the results of the decellularization of tissues and organs for 1 hour when they are treated with various solutions. Table 1 demonstrates that the present invention allows to obtain decellularized organs and tissues when treated with solutions 1-3 for 1 hour, which is not achieved by the comparison solution, the composition of which was taken from patent RU 2653489.

The proposed method is applicable for the decellularization of organs and tissues with good blood supply due to the high density distribution of blood vessels, ducts or interstitial channels in the tissue or organ (liver, kidney, omentum, lungs), which makes it possible to uniformly effect the decellularizing solution in volume, thereby ensuring complete decellularization of the tissue or organ. In addition to whole organs, tissue sections with a high degree of vascularization, for example, cardiac pericardium and proximal tracheal segments, can be used as resection material.

Experiments were conducted on the decellularization of organs such as rat liver, rabbit trachea, rat artery, in which it was possible to reduce the time of decellularization to 70 minutes.

Also conducted experiments with the human bile duct. Due to morphological similarities, the proposed method can be extended not only to biological tissues or organs of a laboratory animal, but also biological tissues or human organs.

Obtained using the proposed method, decellularized organs and tissues can be used in tissue engineering to create autologous transplants, to replace or replace prosthetics of affected organs and tissues.

Example 2. The use of decellularized organs as a component of tissue-engineering design in an experiment on rabbits.

The decellularized trachea of rabbits (n = 3) obtained by the proposed method were used to create tissue-engineering structures of the trachea and were implanted into rabbits in an orthotopic position. When creating a tissue engineering construct, decellularized trachea of rabbits under sterile conditions was populated with nasal chondrocytes, which were cultured on a matrix in a CO ₂ incubator for 1 week in a complete nutrient medium consisting of DMEM / F12 (Invitrogen, USA) containing 10% FBS (Invitrogen , USA), 0.4 μM insulin, 20 ng / ml bFGF, 10 nM dexamethasone, 100 u / ml penicillin and 100 μg / ml streptomycin (Invitrogen, USA). Cells were seeded in an amount of 10 ⁶ cells / ml.

A rabbit fragment was removed from the rabbits under operating conditions and a tissue-engineering construct was installed in the defect area (Fig. 2). Rabbits were monitored, and subsequently histological examination of autopsy material was performed (Fig. 3).

Decellularized material obtained from rabbit trachea by an accelerated method of treatment with a solution consisting of (wt.%) 1.7-4.1% hydroxymethylaminomethane (Tris), 4.62-11.62% sodium chloride (NaCl), 0.08 -0.16% sodium dodecyl sulfate (SDS), 0.25-0.65% sodium deoxycholate (SDC), 0.65-1.25% Triton X-100, in deionized water - was suitable for creating a tissue-engineering design of the trachea and after implantation, the rabbit performed a biological function for a long observation period (6 months of observation).

Claims (3)

1. The method of decellularization of a biological tissue or organ, characterized in that it involves the removal of cellular elements by washing the biological tissue or organ with a solution containing tris (hydroxymethyl) aminomethane, sodium chloride, sodium dodecyl sulfate, sodium deoxycholate, Triton X-100, and deionized water at the following components, wt. %: Tris (hydroxymethyl) aminomethane 1.7-4.1 Sodium chloride 4.62-11.62 Sodium dodecyl sulfate 0.08-0.16 Sodium deoxycholate 0.25-0.65 Triton X-100 0.65-1.25 Deionized water up to 100 2. The method according to p. 1, characterized in that the biological tissue or organ is selected from the group consisting of the liver, blood vessel, trachea, lung, heart, intestines, stomach and bile duct.

Images