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 Federation10.15825/1995-1191-2022-2-83-93vtio-1500Research ArticleРегенеративная медицина и клеточные технологииRegenerative Medicine and Cell TechnologiesКриогенно-структурированный гидрогель на основе желатина как резорбируемая макропористая матрица для биомедицинских технологийCryogenically structured gelatin-based hydrogel as a resorbable macroporous matrix for biomedical technologiesГригорьевА. М.GrigorievA. M.

Григорьев Алексей Михайлович

123182, Москва, ул. Щукинская, д. 1Тел. (499) 193-86-62

Alexey Grigoryev

1, Shchukinskaya str., Moscow, 123182Phone: (499) 193-86-62

Bear-38@yandex.ruБасокЮ. Б.BasokYu. B.

Москва

Moscow

bjb2005@mail.ruКирилловаА. Д.KirillovaA. D.

Москва

Moscow

kirillovaad20@gmail.comСургученкоВ. А.SurguchenkoV. A.

Москва

Moscow

valent.surg@yandex.ruШмеркоН. П.ShmerkoN. P.

Москва

Moscow

shnape@mail.ruКулаковаВ. К.KulakovaV. K.

Москва

Moscow

valkirkul@mail.ruИвановР. В.IvanovR. V.

Москва

Moscow

erawell@mail.ruЛозинскийВ. И.LozinskyV. I.

Москва

Moscow

loz@ineos.ac.ruСубботА. М.SubbotA. M.

Москва

Moscow

kletkagb@gmail.comСевастьяновВ. 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 FederationФГБУН «Институт элементоорганических соединений имени А.Н. Несмеянова» Российской академии наукРоссияNesmeyanov Institute of Organoelement CompoundsRussian FederationФГБНУ «Научно-исследовательский институт глазных болезней»РоссияResearch Institute of Eye DiseasesRussian Federation2022130520222428393Copyright © Григорьев А.М., Басок Ю.Б., Кириллова А.Д., Сургученко В.А., Шмерко Н.П., Кулакова В.К., Иванов Р.В., Лозинский В.И., Суббот А.М., Севастьянов В.И., 20222022Григорьев А.М., Басок Ю.Б., Кириллова А.Д., Сургученко В.А., Шмерко Н.П., Кулакова В.К., Иванов Р.В., Лозинский В.И., Суббот А.М., Севастьянов В.И.Grigoriev A.M., Basok Y.B., Kirillova A.D., Surguchenko V.A., Shmerko N.P., Kulakova V.K., Ivanov R.V., Lozinsky V.I., Subbot A.M., 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/1500

Цель: исследование биологических свойств матрицы из криогенно-структурированного гидрогеля в форме макропористой желатиновой губки, а также возможности создания на ее основе клеточно-инженерных конструкций. Материалы и методы. Основными компонентами криогенно-структурированного гидрогеля были желатин (тип А), полученный из коллагена свиной кожи, N-(3-диметиламинопропил)-N’-этил-карбодиимид, (ЭДК) и мочевина (все – Sigma-Aldrich, США). Морфологию поверхности исследовали с использованием сканирующей электронной микроскопии. Степень набухания в воде образцов определяли гравиметрическим методом. Цитотоксичность исследовали на фибробластах мыши линии NIH 3T3 и мезенхимальных стромальных клетках жировой ткани человека (МСК ЖТч) с использованием IncuCyte ZOOM (EssenBioscience, США). Метаболическую активность МСК ЖТч оценивали с помощью реагентов PrestoBlue™ (Invitrogen™, США). Для создания клеточно-инженерных конструкций (КИК) использовали МСК ЖТч, клетки гепатоцеллюлярной карциномы HepG2 или эндотелиальные клетки пупочной вены человека линии EA.hy926. Содержание альбумина в культуральной среде определяли методом иммуноферментного анализа. Скорость метаболизма аммиака оценивали после 90 минут инкубации с 1 mM хлоридом аммония (Sigma-Aldrich, США), разведенным в культуральной среде на 15-е сутки эксперимента. Результаты. Получение матрицы из криогенно-структурированного гидрогеля в форме макропористой желатиновой губки включало замораживание водного раствора смеси желатина и мочевины, удаление поликристаллов замерзшего растворителя лиофилизацией, экстракцию мочевины этанолом и обработку криоструктурата этанольным раствором ЭДК. Сканирующая электронная микроскопия позволила выделить три типа пор на поверхности носителя: крупные (109 ± 17 мкм), средние (39 ± 10 мкм) и малые (16 ± 6 мкм). Степень набухания в воде образцов матрицы составила 3,8 ± 0,2 г Н2О на 1 г сухого полимера. Установлена способность макропористой желатиновой губки в составе КИК поддерживать адгезию и пролиферацию МСК ЖТч, эндотелиальных клеток пупочной вены человека линии EA.hy926 и HepG2 в течение 28, 15 и 9 суток соответственно. Доказано наличие секреции альбумина и метаболизма аммиака при культивировании клеток HepG2 на желатиновой губке. Заключение. На примере клеточно-инженерной конструкции печени показана перспективность использования матрицы из макропористого криогенно-структурированного гидрогеля на основе желатина для создания продуктов тканевой инженерии.

Objective: to investigate the biological properties of a matrix made of cryogenically structured hydrogel in the form of a macroporous gelatin sponge, as well as the possibility of creating cell-engineered constructs (CECs) on its basis. Materials and methods. The main components of the cryogenically structured hydrogel were gelatin (type A) obtained from porcine skin collagen, N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide, (EDC) and urea (all from Sigma-Aldrich, USA). Surface morphology was examined using scanning electron microscopy (SEM). The degree of swelling in water of the samples was determined by gravimetric method. Cytotoxicity was studied on NIH3T3, a fibroblast cell line isolated from a mouse, and on human adipose-derived mesenchymal stem/stromal cells (hAMSCs) using IncuCyte ZOOM (EssenBioscience, USA). The metabolic activity of hAMSCs was assessed using PrestoBlue™ reagents (Invitrogen™, USA). To create CECs, we used hAMSCs, human hepatocellular carcinoma cell line HepG2 or human umbilical vein endothelial cell lines EA.hy926. Albumin content in the culture medium was determined by enzyme immunoassay. Ammonia metabolism rate was assessed after 90 minutes of incubation with 1 mM ammonium chloride (Sigma-Aldrich, USA) diluted in a culture medium on day 15 of the experiment. Results. Obtaining a cryogenically structured hydrogel scaffold in the form of macroporous gelatin sponge included freezing an aqueous solution of a gelatin+urea mixture, removal of polycrystals of frozen solvent by lyophilization, extraction of urea with ethanol and treatment of the cryostructurate with an ethanol solution of EDC. Scanning electron microscopy identified three types of pores on the carrier surface: large (109 ± 17 μm), medium (39 ± 10 μm), and small (16 ± 6 μm). The degree of swelling in water of the matrix samples was 3.8 ± 0.2 g H2O per 1 g of dry polymer. The macroporous gelatin sponge as a part of CEC was found to have the ability to support adhesion and proliferation of hAMSCs, EA.hy926 and HepG2 for 28, 15 and 9 days, respectively. Albumin secretion and ammonia metabolism when HepG2 cells were cultured on the gelatin sponge were detected. Conclusion. The use of a matrix made from macroporous cryogenically structured gelatin-based hydrogel for tissue engineering products is shown to be promising using a cell-engineered liver construct as a case.

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