Модель биомедицинского клеточного продукта для доклинических исследований на крупном лабораторном животном

Objective: to develop a model of a biomedical cell product that is consistent with the «homologous drug» strategy  based on protocols for preparing the cell component and scaffold carrier for preclinical studies on a large laboratory  animal (pig). Materials and methods. Biomedical cell products and skin equivalents (SE), were formed using  plasma cryoprecipitate prepared from blood plasma of healthy donors and mesenchymal stem cells (MSCs) of  human adipose tissue. Cryoprecipitate from pig blood plasma and human adipose tissue-derived MSCs were used   to form model skin equivalents (mSE). Bright-field microscopy, phase-contrast microscopy (Leica DMI 3000B)  and fluorescence microscopy (Cytation 5 imager; BioTek, USA) were used to monitor the state of cells in the  culture and in the composition of the equivalents. Scaffolds for equivalents were tested for cytotoxicity (MTT test,  direct contact method). The cell distribution density was characterized by author’s method (Patent No. 2675376  of the Russian Federation). Results. An mSE was developed for preclinical studies on a large laboratory animal  (pig). In the mSE, components that change from halogen to xenogenic conditions during transplantation to the  animal were replaced. A comprehensive approach to preparing mSE was presented. It includes sampling of primary  pig biomaterial, extraction and characterization of adipose tissue-derived MSCs, preparation of a scaffold  carrier for the corresponding «homologous drug» strategy. Cytotoxicity of the mSE scaffold was evaluated. It  was shown that mSE provides mechanical support (similar to SE) to cells, as well as comparable development of  cellular events during cultivation. Conclusion. A model of a biomedical cell product was developed. This model  is consistent with the «homologous drug» strategy for preclinical studies on a large laboratory animal (pig). The  paper presented a comprehensive approach to developing a model equivalent based on protocols for preparation  and testing of the cellular component, the scaffold carrier and the ready-to-use model equivalent.Цель: разработать модель биомедицинского клеточного продукта, согласующуюся со стратегией «гомологичный препарат» на основе протоколов подготовки клеточной составляющей и скаффолда-носителя для доклинических исследований на крупном лабораторном животном (свинье). Материалы и методы. Биомедицинские клеточные продукты – эквиваленты кожи (ЭК) формировали с использованием криопреципитата плазмы крови здоровых доноров и мезенхимальных стволовых клеток (MSCs) жировой ткани человека. Для формирования модельных эквивалентов кожи (мЭК) использовали криопреципитат плазмы крови свиней и MSCs жировой ткани свиней. Наблюдение за состоянием клеток в культуре и в составе эквивалентов проводили с использованием методов светлого поля, фазового контраста (Leica DMI 3000B) и флуоресцентной микроскопии (имиджер Cytation 5; BioTek, USA). Скаффолды эквивалентов тестировали на цитотоксичность (МТТ-тест, метод прямого контакта). Характеристику плотности распределения клеток проводили авторским способом (Пат. № 2675376 РФ). Результаты. Разработан модельный эквивалент кожи (мЭК) для проведения доклинических исследований на крупном лабораторном животном (свинье). В мЭК замещены компоненты, переходящие из алогенных условий в ксеногенные при трансплантации животному. Представлен комплексный подход для подготовки мЭК, включающий забор первичного биоматериала свиньи, выделение и характеристику MSCs жировой ткани, подготовку скаффолда-носителя, соответствующего стратегии «гомологичный препарат». Проведена оценка цитотоксичности скаффолда мЭК. Показано, что мЭК обеспечивает аналогичную эквиваленту кожи (ЭК) механическую поддержку клеток и сопоставимое развитие клеточных событий при культивировании. Вывод. Разработана модель биомедицинского клеточного продукта, согласующаяся со стратегией «гомологичный препарат» для доклинических исследований на крупном лабораторном животном (свинье). Представлен комплексный подход, для разработки модельного эквивалента основанный на протоколах подготовки и тестирования клеточной составляющей, скаффолда-носителя и готового модельного эквивалента

Specific His(6)-tag Attachment to Metal-Functionalized Polymersomes Relies on Molecular Recognition

The development of nanocarriers for drug/protein delivery is in focus today, as they can serve to both decrease dosages and improve localization to a desired biological compartment A powerful tool to functionalize these carriers is specific affinity tagging supported by molecular recognition, a key principle in biology. However, the geometry of the binding region in a molecular recognition process, and thus its conformation and specificity, are in many cases poorly understood. Here, we demonstrate that short, model peptides, His(6)-tags, selectively recognize Cu-II-trisnitrilotriacetic acid moieties (Cu-II-trisNTA) when exposed at the surfaces of polymer vesicles designed to serve as nanocarriers or as surfaces for proteins. binding. A Mixture of poly(butadiene)-b-poly(ethylene oxide) (PB-b-PEO) and Cu-II-trisNTA-functionalized PB-b-PEO diblock copolymers (10:1) self-assembles in aqueous solution, generating vesicles with a hydrodynamic radius of approximately 100 nm, as established by light scattering and TEM. Fluorescently labeled His(6) tags specifically bind to metal centers exposed on vesicles surface, with a dissociation constant of 0.6 +/- 0.2 mu M, as determined by fluorescence correlation spectroscopy. The significant rearrangement in the geometry of the metal center upon peptide binding was characterized by a combination of,CW-EPR, pulse-EPR, and DFT computations. Understanding the binding configuration around the metal center inside NTA pocket exposed at the surface of vesicles supports further. development of efficient targetable nanocarriers that can be recognized selectively by molecular recognition in a biological environment and facilitates their immobilization on solid supports and their use in two-dimensional protein arrays

Biomedical cell product model for preclinical studies carried out on a large laboratory animal

Objective: to develop a model of a biomedical cell product that is consistent with the «homologous drug» strategy  based on protocols for preparing the cell component and scaffold carrier for preclinical studies on a large laboratory  animal (pig). Materials and methods. Biomedical cell products and skin equivalents (SE), were formed using  plasma cryoprecipitate prepared from blood plasma of healthy donors and mesenchymal stem cells (MSCs) of  human adipose tissue. Cryoprecipitate from pig blood plasma and human adipose tissue-derived MSCs were used   to form model skin equivalents (mSE). Bright-field microscopy, phase-contrast microscopy (Leica DMI 3000B)  and fluorescence microscopy (Cytation 5 imager; BioTek, USA) were used to monitor the state of cells in the  culture and in the composition of the equivalents. Scaffolds for equivalents were tested for cytotoxicity (MTT test,  direct contact method). The cell distribution density was characterized by author’s method (Patent No. 2675376  of the Russian Federation). Results. An mSE was developed for preclinical studies on a large laboratory animal  (pig). In the mSE, components that change from halogen to xenogenic conditions during transplantation to the  animal were replaced. A comprehensive approach to preparing mSE was presented. It includes sampling of primary  pig biomaterial, extraction and characterization of adipose tissue-derived MSCs, preparation of a scaffold  carrier for the corresponding «homologous drug» strategy. Cytotoxicity of the mSE scaffold was evaluated. It  was shown that mSE provides mechanical support (similar to SE) to cells, as well as comparable development of  cellular events during cultivation. Conclusion. A model of a biomedical cell product was developed. This model  is consistent with the «homologous drug» strategy for preclinical studies on a large laboratory animal (pig). The  paper presented a comprehensive approach to developing a model equivalent based on protocols for preparation  and testing of the cellular component, the scaffold carrier and the ready-to-use model equivalent