Significance of nutrient media choice for the long-term cultures of leukemic T-lymphoblasts

Correct choice of nutrient media for culturing different types of cells in various applications is one of the most important aspects of modern biotechnology, since chemical composition of the culture media largely contains the necessary metabolites to support certain cells’ growth lines outside the body. Jurkat line of human leukemic T-lymphoblast-like cells (hereinafter Jurkat T-cells) is actively used for in vitro modeling of intracellular signaling and activation of normal blood T-lymphocytes mediated by the T-cell receptor/CD3/ CD4 complex in toxicological studies of immune and secretory responses, to test medicinal substances and ions. Also, Jurkat T-cells are widely used for ex vivo testing in immunology, oncology, toxicology, orthopedics, and traumatology. The existing standards and numerous studies are mainly based on short-term in vitro cultivation of Jurkat T-cells in RPMI 1640 nutrient medium. Meanwhile, the issues of long-term maintenance of the growth of Jurkat T-cells culture are poorly presented in the research literature. This study aimed for studying the activity of Jurkat T-cells over 7 to 14 days of in vitro culture and comparing the relative value of RPMI 1640 and αMEM media for the behavior of immunocompetent tumor cells. Using flow cytometry, multiplex analysis, and phase contrast Cell-IQ microscopy, the proportions of living cells and those dying by apoptosis and necrosis, secretion of cytokines and chemokines, and the dynamics of cell biomass propagation were studied. It was found that the αMEM medium in the complete nutrient medium, as compared with RPMI 1640, is more appropriate to in vitro promotion of cell viability (increased proportion of viable cells by 13.5% at the day 14), their secretory ability for 23 из 27 tested biomolecules, shortened adaptation time (на 32%) in culture before growth initiation, 5-fold increase of the Jurkat Т-cell cellularity by the day 7. Potential significance of the chemical components of nutrient media and secreted biomolecules for these results is discussed. As based on the results obtained, we concluded on superior properties of αMEM medium for long-term in vitro cultures of Jurkat T-cells. Consequently, the in vitro testing of medical devices intended for long-term contact with the body, including those for cancer patients, using Jurkat T-cell leukemia line in RPMI 1640 medium, may lead to wrong predictions on their biocompatibility and potential antitumor activity

Моделирование микроокружения мезенхимных стволовых клеток как перспективный подход к тканевой инженерии и регенеративной медицине (краткий обзор)

One of the promising areas is the design and modification of materials for control over the fate of multipotent mesenchymal stromal cells (MMSCs) that will allow stroma of various human and animal organs and tissues to be constructed. However, the discussion about the existence and functioning of microenvironment for the MMSCs is just beginning to develop. The design of artificial materials that are able to reproduce biomimetically the cellular and tissue microenvironment and based on ideas and main elements borrowed from wildlife is current direction in a development of medical materials technology and tissue bioengineering. Scaffold technology is a promising experimental approach to simulate the properties of natural microenvironment of stem cells. Our aim is a short review of key elements of MMSC microterritories, its advanced investigations and the attempts of modeling in application to tissue bioengineering and regenerative medicine.Одним из перспективных направлений являются разработка и модификация материалов для контроля жизнедеятельности мультипотентных мезенхимных стромальных клеток (ММСК), которые (ре)конструируют строму различных органов и тканей человека и животных. Тем не менее обсуждение вопроса о существовании и функционировании микроокружения ММСК только начинается. Это тормозит дальнейшее развитие клеточной биологии и тканевой инженерии. Дизайн искусственных материалов, способных к биомиметическому воспроизведению клеточного и тканевого микроокружения, основанный на идеях и элементах, заимствованных у природы, является современным направлением в развитии медицинского материаловедения и тканевой инженерии. Скеффолд-технологии – многообещающий экспериментальный подход к моделированию свойств природного микроокружения для стволовых клеток. Цель – краткий обзор ключевых элементов микротерриторий ММСК, его перспективных исследований и попыток моделирования в приложении к тканевой инженерии и регенеративной медицине.

Морфофункциональное состояние культуры стволовых клеток на 2D-матриксе, имитирующем «молчащие» остеогенные и кроветворные микротерритории

An influence relief features and quantitative parameters of model bone mineral matrix on in vitro morphofunctional status of human prenatal stromal lung cells (HPSLC) and in vivo mice bone/marrow system remodeling has been studied. Investigations have showed that magnetron calcium phosphate (CaP) coatings are thin (thickness less than 1—2 μm), nonporous, smooth (Ra < 1 μm) layer of carboxylated apatites with the calcium/phosphorus ratio higher than stoichiometric one (more than 1.67). Such 2D surfaces have discreet structure-functional microterritories (niches) for HPSLC. Nevertheless, HPSLC maturation in osteoblasts under 4-days cultivation on such surfaces is unlikely. Poor HPSLC osteogenic committing on «smooth» (Ra < 1 μm) CaP coating is in vitro associated with an average index of artificial osteogenic niche less than 34%, weak artificial surface dissolution and elevated TNFα secretion by cell culture. A behavior of such implants with singenic bone marrow column is displayed in vivo by a degeneration of mice heterotopic osteogenesis and hemopoiesis. Methodology suggested allows apparently to imitate bone surface microterritories having “quiescent“ endosteal niches for stromal and hemopoietic stem cells.Изучено влияние особенностей рельефа и количественных параметров модельного костного минерального матрикса на морфофункциональное состояние пренатальных стромальных клеток легкого человека (ПСКЛЧ) in vitro и ремоделирование системы кость — костный мозг мышей in vivo. Исследования показали, что магнетронные кальцийфосфатные CaP покрытия представляют собой тонкий (толщина не более 1—2 мкм) беспористый гладкий (Ra меньше 1 мкм) слой карбоксилированных апатитов с соотношением кальция и фосфора выше стехиометрического (более 1,67). Такие 2D-поверхности несут дискретные структурно-функциональные микротерритории (ниши) для ПСКЛЧ. Тем не менее созревание ПСКЛЧ в остеобласты при 4-суточном культивировании на подобных поверхностях является маловероятным. Слабое остеогенное коммитирование ПСКЛЧ на гладком (Ra меньше 1 мкм) CaP покрытии ассоциировано in vitro со средним индексом искусственной остеогенной ниши менее 34%, незначительным растворением искусственной поверхности, повышенной секрецией TNF-α в культуре клеток. Поведение подобных имплантатов, несущих столбик сингенного костного мозга, проявляется in vivo затуханием эктопического костеобразования и гемопоэза у мышей. Предложенная методология позволяет, по-видимому, имитировать участки поверхности кости, несущие «молчащие» эндостальные ниши для стромальных и кроветворных стволовых клеток

Pathophysiological and clinical problems of cellular immunotherapy of digestive system cancer. Critical review

The review is devoted to the methods of bioimmunotherapy of oncological pathology used in the clinic for the recent 20—25 years and, to the higher extent, the principles of cellular immunotherapy of cancerous epithelial tumors of the digestive system. The surgical method is recognized as a «gold standard» in the therapy of the digestive system cancer. Bioimmunotherapy of the digestive system cancer has not achieved the sufficient clinical efficiency. In this connection, new pathophysiological approaches to biotherapy of the digestive system cancer based, in particular, on original results of Russian scientists are analyzed

Novel Concepts of “Niche-Relief” and Niche-Voltage” for Stem Cells as a Base of Bone and Hematopoietic Tissues Biomimetic Engineering

An affect of relief features and quantitative variables of artificial surfaces on structural-functional status of human lung prenatal stromal cells (HLPSC) and remodeling of Balb/c mice bone/bone marrow system have been studied. Implants with rough (Ra>2 μm) calcium phosphate micro-arc coatings have structural-functional sites (micro-regions) named “niches-relief” which are necessary for maturation and differentiation of HLPSC into secreting osteoblasts in shortterm culture. Maximal remodeling of mouse bone/bone marrow system in 45-day subcutaneous heterotopic test in vivo is also noted under optimal parameter (average index of cellular alkaline phosphatase area to artificial micro-region area is about 43 %) of osteogenic niche in vitro. Probable physical mechanism of osteogenic niche functioning has been determined. It correlates with increasing of electron work function supplied with a negative charge of calcium phosphate nanorelief in the sockets (artificial “niches”). “Niche-voltage” concept for bone tissue biomimetic engineering was proposed

Novel Concepts of “Niche-Relief” and Niche-Voltage” for Stem Cells as a Base of Bone and Hematopoietic Tissues Biomimetic Engineering

Currently, a lot of publications, mainly as hypotheses, were dedicated to niches for hemopoietic stem cells (HSC). Multipotent mesenchymal stromal cells (MMSC) are treated as important cell component of HSC niches [1]. At the same time, the circumstance that discussion of existence of niches determining fate of MMSC themselves just appears in literature sets at gaze [2]. Pilot experimental data about existence and dimensions of niches for osteogenic differentiation of MMSC, their close dependence on quantitative variables of bone mineral substance relief were recently obtained by us [3]. In this connection, investigation of the relief features effects of model mineral matrix on structural-functional conditions of human MMSC and remodeling of bone/bone marrow system in mice was of great interest

Morphofunctional status of stem cells culture on 2D matrix imitating «quiescent» osteogenic and hemopoietic microterritories

An influence relief features and quantitative parameters of model bone mineral matrix on in vitro morphofunctional status of human prenatal stromal lung cells (HPSLC) and in vivo mice bone/marrow system remodeling has been studied. Investigations have showed that magnetron calcium phosphate (CaP) coatings are thin (thickness less than 1—2 μm), nonporous, smooth (Ra < 1 μm) layer of carboxylated apatites with the calcium/phosphorus ratio higher than stoichiometric one (more than 1.67). Such 2D surfaces have discreet structure-functional microterritories (niches) for HPSLC. Nevertheless, HPSLC maturation in osteoblasts under 4-days cultivation on such surfaces is unlikely. Poor HPSLC osteogenic committing on «smooth» (Ra < 1 μm) CaP coating is in vitro associated with an average index of artificial osteogenic niche less than 34%, weak artificial surface dissolution and elevated TNFα secretion by cell culture. A behavior of such implants with singenic bone marrow column is displayed in vivo by a degeneration of mice heterotopic osteogenesis and hemopoiesis. Methodology suggested allows apparently to imitate bone surface microterritories having “quiescent“ endosteal niches for stromal and hemopoietic stem cells

Modeling of the mesenchymal stem cell microenvironment as a prospective approach to tissue bioengineering and regenerative medicine (a short review)

One of the promising areas is the design and modification of materials for control over the fate of multipotent mesenchymal stromal cells (MMSCs) that will allow stroma of various human and animal organs and tissues to be constructed. However, the discussion about the existence and functioning of microenvironment for the MMSCs is just beginning to develop. The design of artificial materials that are able to reproduce biomimetically the cellular and tissue microenvironment and based on ideas and main elements borrowed from wildlife is current direction in a development of medical materials technology and tissue bioengineering. Scaffold technology is a promising experimental approach to simulate the properties of natural microenvironment of stem cells. Our aim is a short review of key elements of MMSC microterritories, its advanced investigations and the attempts of modeling in application to tissue bioengineering and regenerative medicine