mesЭкстремальная биомедицинаExtreme Medicine3033-89643033-8972Centre for Strategic Planning of the Federal Medical and Biological Agency10.47183/mes.2025-27-1-97-106mes-280Research ArticleРЕГЕНЕРАТИВНАЯ МЕДИЦИНАREGENERATIVE MEDICINEОпределение состава протеогликанов, синтезируемых in vitro хондроцитами различного генезаComposition analysis of proteoglycans synthesized in vitro by chondrocytes of various originshttps://orcid.org/0000-0002-7414-7326ГолубинскаяП. А.GolubinskayaP. A.

Голубинская Полина Александровна - канд. мед. наук

Москва

Polina A. Golubinskaya

Moscow

polinapigeon@gmail.comhttps://orcid.org/0000-0002-1361-666XРучкоЕ. С.RuchkoE. S.

Ручко Евгений Сергеевич

Москва

Evgeny S. Ruchko

Moscow

ruchkoevgeny@yandex.ruhttps://orcid.org/0000-0002-8967-2318ПикинаА. С.PikinaA. S.

Пикина Арина Сергеевна

Москва

Arina S. Pikina

Moscow

arina.pikina@yandex.ruhttps://orcid.org/0000-0003-0402-3392СмирновИ. П.SmirnovI. P.

Смирнов Игорь Павлович - канд. хим. наук

Москва

Igor P. Smirnov

Moscow

smirnov_i@hotmail.comhttps://orcid.org/0009-0001-9266-2065ВладимироваТ. В.VladimirovaT. V.

Владимирова Татьяна Викторовна

Москва

Tatiana V. Vladimirova

Moscow

tat.vlad24@gmail.comhttps://orcid.org/0000-0001-8904-8235ГордееваВ. Д.GordeevaV. D.

Гордеева Вероника Дмитриевна - канд. биол. наук

Москва

Veronika D. Gordeeva

Moscow

gordeeva.veronika@phystech.eduhttps://orcid.org/0000-0003-2323-1859АрапидиГ. П.ArapidiG. P.

Арапиди Георгий Павлович - канд. биол. наук

Москва

Georgiy P. Arapidi

Moscow

arapidi@gmail.comhttps://orcid.org/0000-0002-3428-7586ЕремеевА. В.EremeevA. V.

Еремеев Артем Валерьевич - канд. биол. наук

Москва

Artem V. Eremeev

Moscow

art-eremeev@yandex.ruФедеральный научно-клинический центр физико-химической медицины имени Ю.М. Лопухина Федерального медико-биологического агентстваРоссияLopukhin Federal Research and Clinical Center of Physical-Chemical MedicineRussian Federation20252403202527197106Copyright © Голубинская П.А., Ручко Е.С., Пикина А.С., Смирнов И.П., Владимирова Т.В., Гордеева В.Д., Арапиди Г.П., Еремеев А.В., 20252025Голубинская П.А., Ручко Е.С., Пикина А.С., Смирнов И.П., Владимирова Т.В., Гордеева В.Д., Арапиди Г.П., Еремеев А.В.Golubinskaya P.A., Ruchko E.S., Pikina A.S., Smirnov I.P., Vladimirova T.V., Gordeeva V.D., Arapidi G.P., Eremeev A.V.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.extrememedicine.ru/jour/article/view/280Введение

Введение. Применение клеточных конструкций на основе плюрипотентных стволовых клеток человека (чПСК) связано с рядом трудностей, одной из них является необходимость стандартизации методов культивирования хондроцитоподобных производных чПСК для получения хрящевой ткани, схожей с естественным гиалиновым хрящом. Гликозаминогликаны (ГАГ) – основа внеклеточного матрикса (ВКМ) хрящевой ткани, поэтому анализ качественного и количественного состава ГАГ хрящеподобных тканеинженерных конструкций является важным звеном для итоговой оценки их потенциальной терапевтической эффективности.

Цель

Цель. Определить состав ГАГ, синтезируемых in vitro хондроцитами различного генеза, с использованием иммуноферментного анализа (ИФА) и жидкостной хроматографии с тандемной масс-спектрометрией (ЖХ-МС/МС), а также оценить влияние 2D- и 3D-культивирования на их синтез.

Материалы и методы

Материалы и методы. В исследовании анализировали уровни ГАГ в 2D- и 3D-тканеинженерных конструкциях, полученных из хрящевой ткани 5 доноров, хондроцитоподобных клеток, дифференцированных из 2-х линий чПСК. Клеточные сфероиды получали методом агрегации в микролуночных планшетах и культивировали в мини-биореакторах. Анализ содержания ГАГ в образцах клеточных культур и сфероидов проведен с помощью ИФА и ЖХ-МС/МС. Для оценки статистической значимости различий между образцами использовали тест Крускала-Уоллиса и тест Данна.

Результаты

Результаты. В исследовании с помощью ИФА выявлены статистически значимые различия (p<0,0021), подтверждающие более высокий уровень синтеза ГАГ в 3D-культурах нативных хондроцитов по сравнению с 2D-культурами (108,67 нг/мл и 1099,87 нг/мл соответственно). Среднее число спектров белка хондроитинсульфат-протеогликана 4, определенное с помощью ЖХ-МС/МС, также было выше в 3D-культурах, составив 41,75 спектров по сравнению с 2,24 спектров в образцах 2D-культур. Уровни аггрекана, бигликана и декорина между культурами не различались. 3D-культуры хондроцитоподобных клеток из чПСК не показали достоверных отличий в содержании ГАГ по сравнению с 2D, что указывает на необходимость оптимизации условий их дифференцировки.

Выводы

Выводы. В нашем исследовании был определен состав ГАГ, синтезируемых in vitro хондроцитами различного генеза, с использованием ИФА и ЖХ-МС/МС, а также оценено влияние 2D- и 3D-культивирования на их синтез. Результаты показали, что 3D-среда культивирования создает условия, способствующие более полноценному формированию хондроцитарного ВКМ в образцах нативных хондроцитов. Однако, несмотря на это, полученные сфероиды хондроцитоподобных производных чПСК не достигают функциональной идентичности с естественной хрящевой тканью, даже после завершения протоколов дифференцировки, и не представляют собой идеальные тканеинженерные конструкции для коррекции дефектов хряща.

Introduction

Introduction. The use of cellular constructs based on human pluripotent stem cells (hPSCs) is associated with a number of challenges, including the need to standardize methods for cultivating chondrocyte-like hPSCs derivatives to produce a cartilage tissue similar to natural hyaline cartilage. Glycosaminoglycans (GAGs) are the basis of the extracellular matrix (ECM) of cartilage tissue; therefore, a qualitative and quantitative analysis of the GAG composition of cartilage-like tissue engineering structures is an important step in the final assessment of their potential therapeutic effectiveness.

Objective

Objective. To determine the composition of GAGs synthesized in vitro by chondrocytes of various origins using enzyme-linked immunoassay (ELISA) and liquid chromatography with tandem mass spectrometry (LC–MS/MS), as well as to evaluate the effect of 2D and 3D culturing on their synthesis.

Materials and methods

Materials and methods. We analyzed the GAG levels in 2D and 3D tissue-engineered structures obtained from the cartilage tissue of five donors, chondrocyte-like cells differentiated from two hPSC lines. Cellular spheroids were obtained by aggregation in microlunar plates and cultured in mini-bioreactors. The analysis of the GAG content in cell culture samples and spheroids was carried out using ELISA and LC–MS/MS. The Kruskal–Wallis and Dunn tests were used to assess the statistical significance of the differences between the samples.

Results

Results. The ELISA study revealed statistically significant differences (p < 0.0021), confirming higher levels of GAGs synthesized in 3D cultures of native chondrocytes compared to 2D cultures (108.67 ng/mL and 1099.87 ng/mL, respectively). The average number of spectra of the chondroitin sulfate proteoglycan 4 protein, determined using LC–MS/MS, was also higher in 3D cultures, amounting to 41.75 spectra compared to 2.24 spectra in 2D culture samples. The levels of aggrecan, biglican, and decorin did not differ between cultures. 3D cultures of chondrocyte-like cells from hPSC showed no significant differences in the content of GAG compared to 2D cultures, which indicates the need to optimize the conditions for their differentiation.

Conclusions

Conclusions. In our study, the composition of the GAGs synthesized in vitro by chondrocytes of various origins was determined using ELISA and LC–MS/MS. The the effect of 2D and 3D cultivation on their synthesis was evaluated. The results showed that 3D culture media create favorable conditions for a more complete formation of chondrocytic ECM in native chondrocyte samples. Despite this, the obtained spheroids of chondrocyte-like hPSCs derivatives fail to achieve functional identity with natural cartilage tissue, even after completion of differentiation protocols, thus not representing optimal tissue engineering structures for correcting cartilage defects.

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