vtioВестник трансплантологии и искусственных органовRussian Journal of Transplantology and Artificial Organs1995-1191Academician V.I.Shumakov National Medical Research Center of Transplantology and Artificial Organs", Ministry of Health of the Russian Federation10.15825/1995-1191-2025-1-103-113vtio-1869Research ArticleРегенеративная медицина и клеточные технологииRegenerative Medicine and Cell TechnologiesХимическая децеллюляризация ткани печени свиньи путем двухэтапной обработки поверхностно-активными и осморегулирующими веществами способствует повышению сохранности структуры внеклеточного матрикса печениChemical decellularization of porcine liver by two-stage treatment with surfactants and osmoregulators enhances preservation of liver extracellular matrix structureБеловаА. Д.BelovaA. D.

Белова Александра Дмитриевна 

123182, Москва, ул. Щукинская, д. 1. Тел. (963) 633-94-34 

Aleksandra Belova

1, Shchukinskaya str., Moscow, 123182 Phone: (963) 633-94-34

sashak1994@mail.ruНемецЕ. А.NemetsE. A.

Москва 

Moscow

evgnemets@yandex.ruФилинД. Д.FilinD. D.

Москва 

Moscow

filin_denis@outlook.comПономареваА. С.PonomarevaA. S.

Москва 

Moscow

a.s.ponomareva@gmail.comКирсановаЛ. А.KirsanovaL. A.

Москва 

Moscow

lyudochkakirsanova@mail.ruБасокЮ. Б.BasokYu. B.

Москва 

Moscow

bjb2005@mail.ruСевастьяновВ. И.SevastianovV. I.

Москва 

Moscow

viksev@yandex.ruФГБУ «Национальный медицинский исследовательский центр трансплантологии и искусственных органов имени академика В.И. Шумакова» Минздрава РоссииРоссияShumakov National Medical Research Center of Transplantology and Artificial OrgansRussian FederationФГБУ «Национальный медицинский исследовательский центр трансплантологии и искусственных органов имени академика В.И. Шумакова» Минздрава России ; АНО «Институт медико-биологических исследований и технологий»РоссияShumakov National Medical Research Center of Transplantology and Artificial Organs ; Institute of Biomedical Research and TechnologyRussian Federation202526032025271103113Copyright © Белова А.Д., Немец Е.А., Филин Д.Д., Пономарева А.С., Кирсанова Л.А., Басок Ю.Б., Севастьянов В.И., 20252025Белова А.Д., Немец Е.А., Филин Д.Д., Пономарева А.С., Кирсанова Л.А., Басок Ю.Б., Севастьянов В.И.Belova A.D., Nemets E.A., Filin D.D., Ponomareva A.S., Kirsanova L.A., Basok Y.B., Sevastianov V.I.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://journal.transpl.ru/vtio/article/view/1869

Цель: разработать и исследовать тканеспецифический матрикс, полученный с применением модифицированного режима химической децеллюляризации фрагментов печени свиньи, направленного на эффективное повышение сохранности структуры внеклеточного матрикса (ВКМ), сокращение срока децеллюляризации и улучшение очистки матрикса от клеточных элементов. Материалы и методы. Исходную ткань свиной печени измельчали для получения тканевых фрагментов. Применяли 5 режимов децеллюляризации, последовательно меняя концентрации и сроки обработки поверхностно-активными веществами (ПАВ): 0,1% додецилсульфат натрия (SDS) и 0,1% или 1% Triton X-100, без и в сочетании с фосфатно-солевым буфером (PBS). Содержание гликозаминогликанов (ГАГ) в полученных фрагментах определяли, лизируя образцы 12 ч в растворе папаина при +65 °С с последующей инкубацией в красителе – 1,9-диметилметиленовом синем. Количественное определение ДНК в образцах проводили с применением набора DNeasy Blood&Tissue Kit и красителя Quant-iT PicoGreen. Морфологию образцов исследовали с использованием гистологических методов окрашивания. Цитотоксичность полученных образцов in vitro оценивали на культуре фибробластов мыши линии NIH/3T3 методом прямого контакта. Результаты. Обработка ткани 0,1% SDS в течение 2,5 ч с дополнительной обработкой 1% Triton X-100, содержащим PBS, в течение 21,5 ч (режим 4) способствовала повышению ГАГ до 11,66 ± 0,61 мкг/мг по сравнению с 0,68 ± 0,06 мкг/мг (режим 5). Дополнительная обработка образцов, полученных в режиме 4, ДНКазой I типа позволила снизить содержание ДНК с 99,75 ± 3,93 до 14,93 ± 4,91 нг/мг, что свидетельствовало об эффективном удалении клеточных компонентов. Цитотоксичность этого матрикса не обнаружена. Заключение. Оптимизация режима химической децеллюляризации фрагментов печени свиньи позволила повысить сохранность структур ВКМ, сократить время децеллюляризации и эффективно снизить содержание клеточных элементов. Модифицированный протокол децеллюляризации фрагментов печени свиньи позволил получить нецитотоксичный тканеспецифический матрикс с низкой потенциальной иммуногенностью и более сохранной структурой ВКМ и количеством ГАГ.

Objective: to develop and investigate a tissue-specific matrix obtained using a modified chemical porcine liver decellularization regime in order to effectively increase preservation of extracellular matrix (ECM) structure, reduce decellularization time and improve purification of the ECM from cellular elements. Materials and methods. Original porcine liver was minced to obtain tissue fragments. Five decellularization regimes were used, with the concentrations and timing of surfactant treatments varied: 0.1% sodium dodecyl sulfate (SDS) and 0.1% or 1% Triton X-100, without and in combination with phosphate-buffered saline (PBS). The glycosaminoglycan (GAG) content of the resulting fragments was determined by lysing the samples for 12 hours in papain solution at +65 °C and then incubating them in 1,9-dimethylmethylene blue. DNA quantification was carried out using DNeasy Blood&Tissue Kit and Quant-iT PicoGreen dye. The morphology of the samples was studied using histological staining techniques. Cytotoxicity of the samples in vitro was evaluated on an NIH/3T3 mouse fibroblast culture by direct contact. Results. Treatment with 0.1% SDS for 2.5 hours with additional treatment with 1% Triton X-100 containing PBS for 21.5 hours (regime 4) increased GAG content to 11.66 ± 0.61 μg/mg compared to 0.68 ± 0.06 μg/mg (regime 5). The DNA content of samples obtained in regime 4 decreased from 99.75 ± 3.93 ng/mg to 14.93 ± 4.91 ng/mg after additional treatment with type I DNase, indicating that cellular components were effectively removed. This matrix showed no cytotoxicity. Conclusion. By optimizing the chemical decellularization regime for porcine liver, we were able to improve preservation of ECM structures, shorten decellularization time and effectively reduce the content of cellular elements. The modified decellularization protocol allowed to obtain a non-cytotoxic tissue-specific matrix with a low potential immunogenicity and a more preserved ECM structure and higher GAG content.

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The authors declare that there are no conflicts of interest present.