RU2713803C1 - Method of lung decellularisation for producing extracellular matrix - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to regenerative medicine, and can be used to produce extracellular matrices of organs and tissues used in reconstructive surgery to replace or maintain functions of involved organs and tissues. A method of decellularisation of a rat lung for producing an extracellular matrix (ECM) involves four freezing cycles at -80 °C and successive rinsing of the right and left lungs through trachea with 2.5 ml of deionised water 6–8 times, 2 ml of phosphate-saline buffer with 5 % penicillin and streptomycin, 6–8 times and 5 ml of 1 % sodium dodecyl sulphate. Defrosting is performed at 37 °C. Then, on the second day, the sample is placed in 150 ml of 1 % sodium dodecyl sulphate solution with 0.5 % ethylenediaminetetraacetic acid, at 37 °C and continuous stirring, after which solution of phosphate-salt buffer with 5 % penicillin and streptomycin at 4 °C, where the sample is stored for two weeks before use.

EFFECT: method provides maximum effective removal of cellular structures from lung tissues, while maintaining integrity of the general structure of ECM, reducing the risk of its contamination with microorganisms by washing and using solutions of antibiotics.

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Description

The invention relates to medicine, namely to regenerative medicine, and can be used to obtain extracellular matrices of organs and tissues used in reconstructive surgery to replace or maintain the functions of affected organs and tissues.

The extracellular matrix (VKM), which is used in tissue engineering, can be obtained by various methods of decellularization. Known methods of decellularization involve sequential multi-stage processing of tissue with solutions of detergents and / or enzymes / 1, Hoshiba, T., Lu, N., Kawazoe, N. and Chen, G. (2010). Decellularized matrices for tissue engineering. Expert Opinion on Biological Therapy, 10 (12), pp. 1717-1728. doi: 10.1517 / 14712598.2010.534079 /. Methods of decellularization vary in duration of exposure and concentration of reagents, as well as methods of their introduction. The most commonly used detergents are sodium dodecyl sulfate, Triton X-100, sodium deoxycholate, and trypsin mixed with ethylenediaminetetraacetic acid (EDTA). Decellurization is carried out according to the following schemes: the tissue used is placed in a container with a reagent, providing constant mixing, or perfusion of the lung with reagents through blood vessels is carried out.

There are various methods for lung decellularization, however, the known methods have several disadvantages: all cells are not removed from the treated tissue, their nuclei remain, or mechanical damage to the integrity of the ECM occurs.

A known method of lung decellulation / 2, see patent RU 2547799 /, which consists in constant, corresponding to physiological parameters, ventilation of the lungs with atmospheric air through the trachea. At the same time, perfusion of the lungs is carried out within 24 hours by sequentially introducing through the pulmonary artery decellulating solutions: phosphate buffer, 1% aqueous solution of sodium deoxycholate, porcine pancreatic DNase I, purified water with an equal duration of exposure time.

The disadvantages of this method are the difficulty of conducting catheterization of the pulmonary artery in small laboratory animals, as well as the need to use high-cost chemicals such as DNase and sodium deoxycholate, which makes it difficult to perform this method.

R. and Oliveira, L. (2014). Mechanical properties of mouse lungs along organ decellularization by sodium dodecyl sulfate. Respiratory Physiology & Neurobiology, 200, pp. 1-5. http://dx.doi.org/10.1016/j.resp.2014.04.008/. Данный способ состоит из отмывания комплекса легкого и трахеи от остатков крови, 4 циклов заморозки при -80°С и разморозки при комнатной тепературе, по 10 минут каждый цикл. После чего последовательно через трахею в легкие вводится 2 мл фосфатно-солевого буфера 6-8 раз, 2,5 мл деионизированной воды 6-8 раз, и 2,5 мл 1% раствора додецил сульфата натрия. Далее комплекс органов помещается на 24 часа, последовательно в 1% додецилсульфата натрия при комнатной температуре, затем в фосфатный буфер при постоянном перемешивании при комнатной температуре. После чего легкие перекладываются в чистый раствор фосфатно-солевого буфера для последующего хранения при 4°С.The prototype is a method for decellularization of lung tissue of a mouse / 3, Nonaka, P., Uriarte, J., Campillo, N., Melo, E., Navajas, D.,

R. and Oliveira, L. (2014). Mechanical properties of mouse lungs along organ decellularization by sodium dodecyl sulfate. Respiratory Physiology & Neurobiology, 200, pp. 1-5. http://dx.doi.org/10.1016/j.resp.2014.04.04.008/. This method consists of washing the lung and trachea complex from blood residues, 4 freezing cycles at -80 ° C and defrosting at room temperature, for 10 minutes each cycle. Then, 2 ml of phosphate-buffered saline 6-8 times, 2.5 ml of deionized water 6-8 times, and 2.5 ml of 1% sodium dodecyl sulfate solution are injected into the lungs through the trachea. Next, the organ complex is placed for 24 hours, sequentially in 1% sodium dodecyl sulfate at room temperature, then in a phosphate buffer with constant stirring at room temperature. Then the lungs are transferred to a clean solution of phosphate-saline buffer for subsequent storage at 4 ° C.

The main disadvantages of this method are the insufficient degree of removal of cells and cell elements from the tissue, the low efficiency of using one agent for decellularization, and a high risk of tissue contamination, due to the duration of the protocol and the rejection of antibiotics.

The technical problem solved by this invention is to create a method that allows the most efficient removal of cellular structures from lung tissue, while maintaining the integrity of the overall structure of the ECM, reducing the risk of its contamination by microorganisms.

The essence of the invention lies in the fact that in the method of lung decellularization to obtain an extracellular matrix, comprising four cycles of freezing at -80 ° C and defrosting at 37 ° C, for 10 minutes each cycle, and sequential washing of the right and left lungs through the trachea with solutions: 2 5 ml of denounced water 6-8 times, 2 ml of phosphate-buffered saline + penicillin / streptomycin 5%, 6-8 times, and 5 ml of 1% sodium dodecyl sulfate solution, followed by placement of the organ complex for 24 hours - the first day, in 150 ml of 1% sodium dodecyl sulfate solution at constant stirring, on the first day the sample is placed in 150 ml of a 1% solution of sodium dodecyl sulfate at 37 ° C, and then on the next 24 hours - the second day, the sample is placed in 150 ml of a 1% solution of sodium dodecyl sulfate + 0.5% ethylenediaminetetraacetic acid, at 37 ° C and constant stirring, after which - into a solution of phosphate-saline buffer + penicillin / streptomycin 5% at 4 ° C, where the sample can be stored for two weeks before use.

The method of decellularization of a complex of two lungs is as follows. Prepare a solution No. 1 - 1% solution of sodium dodecyl sulfate in deionized water, the container is placed in a thermostat at 37 ° C until the sodium dodecyl sulfate is completely dissolved. Prepare a solution No. 2 - 1% solution of sodium dodecyl sulfate + 0.5% EDTA in deionized water, the container is placed in a thermostat at 37 ° C until the substances are completely dissolved. Prepare a solution No. 3 - 150 ml of a solution of phosphate-saline buffer + penicillin / streptomycin 5%. A complex of rat organs consisting of a trachea and both lungs is taken. After which the complex undergoes four cycles of freezing at -80 ° C and defrosting at 37 ° C, for 10 minutes each cycle. Next, the lungs are washed by introducing phosphate-buffered saline + penicillin / streptomycin 5%. through the trachea 6-8 times in 2 ml. The next step, the lungs are washed by introducing 2.5 ml of deionized water 6-8 times through the trachea. After washing, 5 ml of 1% sodium dodecyl sulfate is slowly injected into the lungs through the trachea, the solution should fill the lungs. Next, the complex is placed in 150 ml of a 1% sodium dodecyl sulfate solution for 24 hours (first day), with constant stirring at 37 ° C. After treatment with detergent, the lungs are removed and washed by injecting 15 ml of phosphate-saline buffer through the trachea. The next stage, the organs are placed in a 1% solution of sodium dodecyl sulfate + 0.5% EDTA, for 24 hours (second day), with constant stirring of the solution at 37 ° C. The final stage is the placement of the complex from the lungs and trachea in phosphate-buffered saline + penicillin / streptomycin 5% and at 4 ° C. In this solution, the lungs can be stored for two weeks before use.

The invention was verified by histological analysis using hematoxylin-eosin staining and scanning electron microscopy of the obtained decellularized samples. Hematoxylin / eosin staining by light microscopy shows 99.9% removal of cells and their nuclei from lung tissue. Electron microscopy data demonstrate the preservation of the structure of the lung ECM, without signs of violation of its integrity. There are no signs of bacterial contamination.

The technical result from the use of the method is to obtain a decellularized complex of organs that does not contain cellular elements and nucleic acids, consisting of the trachea, the right and left lungs, which can then be used to prepare 3D biological scaffolds with subsequent cultivation of various adhesive cell cultures.

Claims (1)

A method for lung decellularization to obtain an extracellular matrix, including four freezing cycles at -80 ° C and defrosting at 37 ° C, for 10 minutes each cycle and sequential washing of the right and left lungs through the trachea with solutions: 2.5 ml of deionized water 6-8 times , 2 ml of phosphate-buffered saline + penicillin / streptomycin 5%, 6-8 times and 5 ml of 1% sodium dodecyl sulfate solution, followed by placement of the organ complex for 24 hours - the first day, in 150 ml of 1% sodium dodecyl sulfate solution with constant mixing, characterized in that in the first day 24, the sample is placed in 150 ml of a 1% sodium dodecyl sulfate solution at 37 ° C, and then on the next 24 hours the second day, the sample is placed in 150 ml of a 1% sodium dodecyl sulfate solution + 0.5% ethylenediaminetetraacetic acid, at 37 ° C and constant stirring, after which - into a solution of phosphate-saline buffer + penicillin / streptomycin 5% at 4 ° C, where the sample can be stored for two weeks before use.