[Mitomycin C Treatment of Stromal Layers Enhances the Support of In Vitro Hematopoiesis in Co-Culture Systems].

A study was made of the effect that mitomycin C (MitC) treatment of stromal layers of NIH 3T3 cells expressing Jagged1, a ligand of the Notch receptor, exerts on the growth of hematopoietic Lin(-) mouse bone marrow cells in a co-culture system. MitC treatment of stromal cells significantly increased the number of hematopoietic cells and the frequency of colony-forming cells in stromal co-cultures. Transcriptome analysis of control and MitC-treated stromal cell samples was performed by differential RNA sequencing, and genes downregulated by MitC treatment were predominantly associated with the control of cell proliferation, the cell cycle, chromosome segregation, and DNA metabolism. Induction of key hematopoietic cytokines by MitC was not detected by the transcriptome analysis and was therefore not a main factor in the activation of hematopoiesis on the treated stroma. At the same time, the set of the genes most strongly upregulated by MitC treatment is enriched in the genes for cytokines, growth factors, and cell surface proteins, which presumably contribute to enhanced hematopoiesis support on the MitC-treated stroma. Products of some of these genes have been implicated in expansion of hematopoietic stem/progenitor cells in vitro or in vivo.

[Factors Affecting the Labeling of NIH 3T3 Cells with Magnetic Nanoparticles].

The factors that affect the labeling of NIH 3T3 murine fibroblasts with Fe3O4-based magnetic nanoparticles (MNPs) were studied using MNPs produced by the gas condensation and solution precipitation methods and MNPs surface-modified with 3-aminopropylsilane or L-lysine. The production method, surface modifications, the particle concentration and size, the state of the cell population, and the method of MNP introduction were found to substantially affect the efficiency of MNP binding by cells. In particular, large MNP clusters may occur in MNP suspensions in DMSO, and their disruption by sonication increased the percent yield of magnetically labeled cells. Static incubation of a cell suspension led to a more efficient labeling as compared with continuous agitation. Cells attached to a plastic support could be labeled to a higher degree than cells in suspension, but required substantially longer incubations with MNPs. MNP centrifugation on cell layers (magnetic spinoculation) significantly increased the rate and efficiency of labeling. The stability of magnetic labeling was shown to depend on the MNP dose during labeling. Electron microscopy studies demonstrated that MNPs were associated with the cell surface after 20-min incubation with cells and were mostly in the cell interior after 4-h incubation. The results of the study may be useful for preparation and application of magnetized cell samples.

[Modulation of Luciferase Production in Melanoma Cells in vitro].

Reporter proteins find increasing application in biomedical studies in vitro and in vivo. However, to correctly interpret the results based on their use, it is important to understand whether reporter protein production is modulated in model cells and in what conditions such modulation may occur. Reporter activity was studied in Mel IL melanoma cells transiently transfected with a pCpG vector-based plasmid construct expressing firefly luciferase. Luciferase expression quickly dropped during the first two culture passages, which were followed by a quasi-stable period, when luciferase expression relatively slightly decreased with time. Phases of maximal and minimal luciferase production, which corresponded to the exponential and stationary growth phases, respectively, were observed during batch culture. When the medium was changed, luciferase production was stimulated in the stationary, but not exponential, cell growth phase. Severe hypoxia (0.1% O2) decreased the luciferase amount, suggesting substantial modulation of cell metabolism in total and luciferase production in particular. The targeted drug vemurafenib suppressed the luciferase production in Mel IL cells, whereas DMSO, which is often used as a drug solvent in experiments with cells, stimulated the luciferase production. Based on the results, it was hypothesized that modulation of reporter protein production in mammalian cells reflects the adaptation of intracellular metabolism to external conditions and may be a source of incorrect interpretations of experiments using reporter proteins.

[Cytotoxic Effect of Low-Intensity Infrared Laser Irradiation on Human Melanoma Cells].

Continuous low-intensity laser irradiation (LILI) affects the state of cells in culture, including their proliferation rate. Data collected with various cell models vary significantly, but most studies have reported positive effects of LILI on cell proliferation. The effects of continuous infrared LILI (835 nm) was studied using three independent different melanoma cell lines. The LILI effect was shown to strongly depend on the irradiation dose. Higher doses (230 kJ/m^(2)) significantly suppressed the cell growth. A further increase in LILI dose led to a significant cytotoxic effect, which increased disproportionately quickly with the increasing light intensity. Human mesenchymal stem cells (MSCs) were found to be significantly more resistant to the cytotoxic effect of higher-dose LILI. Importantly, the effects were not due to the difference in culture conditions. Control experiments showed that 15 non-melanoma tumor cell lines were more resistant to LILI than melanoma cells. Selective sensitivity of melanoma cells to LILI in vitro was assumed to provide a basis for LILI-based approaches to melanoma treatment.

[Murine and human hematopoietic progenitor cultures grown on stromal layers expressing Notch ligands].

The ex vivo maintenance and expansion of hematopoietic stem cells and early progenitors is necessary for the successful treatment of hematopoietic and immune diseases. Multiple attempts to improve the expansion of hematopoietic stem cells (HSCs) by their cultivation in the presence of growth factor cocktails have so far failed. Novel approaches aimed at conserving the earliest precursors in their undifferentiated state are needed. These approaches should take into account local regulatory factors that are present in the HSC microenvironment and the three-dimensional architecture of their niche. In the present study, we compared the effects of two Notch ligands, i.e., Jagged1 and DLL1, on murine and human hematopoiesis in vitro. Our observations indicate that the stromal expression of Notch ligands increases the production of both the total and phenotypically early murine and human hematopoietic cells in the co-culture. On one hand, this study demonstrates the similarity of effects of stromal expression of Notch ligands on murine and human hematopoiesis in vitro. On the other hand, our study revealed a number of cell type and ligand-specific variations that are systematically described below. It seems that the effects of SCF cytokine addition on murine hematopoiesis in vitro depend on the stromal context and are oppositely directed for Jagged1 and DLL1.

Analysis of Proliferation of Melanoma Cells and Mesenchymal Stem Cells in Co-Culture and Contribution of Experimental Conditions into Interpretation of the Results.

A series of experiments on co-culturing of Mel IL melanoma cells and mesenhymal stem cells showed that these cells do not influence proliferation of each other, but we observed weaker adhesion of stromal stem cells to plastic in cocultures where with melanoma cells were grown on mesenhymal stem cells feeder. Cell proliferation was also considerably influenced by experimental conditions, which should be taken into account for correct interpretation of obtained results. The principles of experiments on co-culturing of cancer and stromal cells are formulated that take into account the most important factors influencing cell behavior and minimize the probability of artifact results. It was concluded that co-culturing conditions cells significantly affect the experimental results and can be the source of conflicting conclusions on mutual influence of stromal and cancer cells in vitro.

[Homogeneous and heterogeneous 3D melanoma models in vitro].

Growth of malignant tumors occurs in three-dimensional space and depends on a presence of stromal component, which performs critical functions of tumor cell protection and growth support. Therefore, development and analysis of tumor models in 3D cell cultures in vitro, including co-culture systems, presents a significant interest. In this study, the results of 3D culturing of two human melanoma cell lines using the hanging drop method, with or without human mesenchymal stem cells (MSCs), are presented. Melanoma lines were shown to behave differently in 3D cultures; in particular, Mel Cher melanoma cells have the ability to form uniform spheroids within 24 h, whereas MeWo cells under similar conditions failed to form spheroids even after 2 days of culture. However, co-culturing of melanoma cells with MSCs resulted in formation of compact 3D cell spheroids in both cases. Visualization of MeWo cells and MSCs in the mixed spheroids using fluorescent dyes revealed certain clustering of melanoma cells. The observed properties of melanoma cells in homogeneous and heterogeneous spheroids may be used in the complex analysis of results of testing of antimelanoma chemotherapy drugs and evaluation of their therapeutic properties.