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 Federation10.15825/1995-1191-2017-4-78-87vtio-831Research ArticleРегенеративная медицина и клеточные технологииRegenerative Medicine and Cell TechnologiesТрехмерный анализ микро- и наноструктуры биоматериалов и клеток методом сканирующей зондовой крионанотомографииThree-dimensional analysis of micro- and nanostructure of biomaterials and cells by method of scanning probe nanotomographyЕфимовА. Е.EfimovA. E.

Москва.

Moscow.

АгаповаО. И.AgapovaO. I.

Москва.

Moscow.

СафоноваЛ. А.SafonovaL. A.

Москва.

Moscow.

БоброваМ. М.BobrovaM. M.

Москва.

Moscow.

АгаповИ. И.AgapovI. I.

Москва.

Moscow.

igor_agapov@mail.ruЛаборатория бионанотехнологий, ФГБУ «Национальный медицинский исследовательский центр трансплантологии и искусственных органов имени академика В.И. Шумакова» Минздрава России.РоссияLaboratory of Bionanotechnology, V.I. Shumakov National Medical Research Center of Transplantology and Artifi cial Organs of the Ministry of Healthcare of the Russian Federation.Russian FederationЛаборатория бионанотехнологий, ФГБУ «Национальный медицинский исследовательский центр трансплантологии и искусственных органов имени академика В.И. Шумакова» Минздрава России; Биологический факультет МГУ им. М.В. Ломоносова.РоссияLaboratory of Bionanotechnology, V.I. Shumakov National Medical Research Center of Transplantology and Artifi cial Organs of the Ministry of Healthcare of the Russian Federation; Faculty of Biology, M.V. Lomonosov Moscow State University.Russian Federation2017310120181947887Copyright © Ефимов А.Е., Агапова О.И., Сафонова Л.А., Боброва М.М., Агапов И.И., 20182018Ефимов А.Е., Агапова О.И., Сафонова Л.А., Боброва М.М., Агапов И.И.Efimov A.E., Agapova O.I., Safonova L.A., Bobrova M.M., Agapov I.I.This work is licensed under a Creative Commons Attribution 4.0 License.https://journal.transpl.ru/vtio/article/view/831Цель

Цель. Провести трехмерный анализ микро- и наноструктуры и количественных морфологических параметров альгинатных сферических клеточных микроносителей и макроносителей на основе регенерированного шелка, модифицированных микрочастицами межклеточного матрикса децеллюляризованной печени крысы, а также клеток гепатокарциномы человека HepG2, адгезированных на микро- и макроносители.

Материалы и методы

Материалы и методы. Трехмерные пористые матриксы из регенерированного шелка, полученные методом выщелачивания, и сферические альгинатные микроносители, полученные методом инкапсуляции, были витализированы клетками гепатокарциномы человека HepG2. Изучение трехмерной структуры клеток и микро- и макроносителей производилось при температуре –120 °С методом сканирующей зондовой крионанотомографии при помощи экспериментальной установки, объединяющей криоультрамикротом и сканирующий зондовый микроскоп.

Результаты

Результаты. Получены трехмерные нанотомографические реконструкции клеток HepG2, адгезированных на стенку макропоры матрикса из регенерированного шелка и на сферический альгинатный микроноситель. Определены морфологические параметры поверхностей макро- и микроносителей и адгезированных клеток: средняя шероховатость, удельная эффективная площадь и длина автокорреляции. Установлено, что средняя шероховатость поверхности альгинатных микроносителей составляет 76,4 ± 7,5 нм, а средняя шероховатость поверхности стенки макроносителя на основе регенерированного шелка – 133,8 ± 16,2 нм, в то время как средняя шероховатость внешних поверхностей клеток, адгезированных на микро- и макроносители, составляет 118,5 ± 9,0 и 158,8 ± 21,6 нм соответственно. Получены трехмерные реконструкции внутриклеточных компартментов размером от 140 до 500 нм.

Выводы

Выводы. Полученные в результате исследования количественные характеристики морфологии поверхностей клеточных носителей и адгезированных на них клеток позволяют исследовать корреляцию морфологических параметров поверхностей клеточных носителей и их биологические свойства. Использование метода сканирующей зондовой крионанотомографии для трехмерного анализа структуры и характеристик биоматериалов, клеток и биоискусственных клеточных конструкций позволит повысить эффективность разработок по созданию новых клеточно-инженерных конструкций с заданными физико-химическими и биологическими характеристиками для задач тканевой инженерии и регенеративной медицины. 

Aim

Aim: to perform a three-dimensional analysis of micro- and nanosctucture and quantitative morphological parameters of alginate spherical microcarriers and porous regenerated silk macrocarriers modifi ed by microparticles of decellularized rat liver matrix and human hepatoma HepG2 cells adhered to micro- and macro carriers.

Materials and methods

Materials and methods. Three-dimensional porous matrices made from regenerated silk by salt leaching technique and alginate spherical microcarriers fabricated by encapsulation were vitalized by human hepatome HepG2 cells. Study of three-dimensional structure of cells and micro- and macro carriers was carried out at –120 °С by scanning probe cryonanotomography technique with use of experimental setup combining cryoultramicrotome and scanning probe microscope.

Results

Results. Three-dimensional nanotomographical reconstructions of HepG2 cells adhered to macropore wall of regenerated silk macrocarrier and to spherical alginate microcarrier are obtained. Morphological parameters (mean roughness, effective surface area and autocorrelation length) are determined for surfaces of macro and microcarriers and adhered cells. The determined mean roughness of alginate microcarrier surface is 76.4 ± 7.5 nm, while that of surface of macropore wall of regenerated silk macrocarrier is 133.8 ± 16.2 nm. At the same time mean roughness of cells adhered to micro- and macrocarriers are 118.5 ± 9.0 и 158.8 ± 21.6 nm correspondingly. Three-dimensional reconstructions of intracellular compartments with dimensions from 140 to 500 nm are also obtained.

Conclusion

Conclusion. Obtained as a result of study quantitative morphology characteristics of surfaces of cell carriers and adhered cells show signifi cant degree of correlation of morphological parameters of cells and their carriers. Use of scanning probe cryonanotomography technique for three-dimensional analysis of structure and characteristics of biomaterials, cells and bio-artifi cial cellular systems enables to improve effi ciency of development of novel cell-engineered constructions with predicted morphological, physical, chemical and biological characteristics for tasks of tissue engineering and regenerative medicine. 

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