Olga O. Udartseva

Polyelectrolyte microcapsules with entrapped multicellular tumor spheroids as a novel tool to study the effects of photodynamic therapy

In the current study, semi-permeable alginate-oligochitosan microcapsules for multicellular tumor spheroids (MTS) generation were elaborated and tested, to estimate a response of the microencapsulated MTS (MMTS) to photodynamic therapy (PDT). The microcapsules (mean diameter 600 μm) with entrapped human breast adenocarcinoma MCF-7 cells were obtained using an electrostatic bead generator, and MMTS were generated by in vitro long-term cell cultivation. The formed MMTS were incubated in Chlorin e6 photosensitizer solution and then irradiated using 650-nm laser light. The cell viability was measured by MTT-assay in 24 h after irradiation, and histological analysis was performed. The proposed MTS-based model was found to be more resistant to the PDT than the two-dimensional monolayer cell culture model. Thus, MMTS could be considered as a promising three-dimesional in vitro model to estimate the doses of drugs or parameters for PDT in vitro before carrying out preclinical tests.

Acute Hypoxic Stress Affects Migration Machinery of Tissue O2-Adapted Adipose Stromal Cells

The ability of mesenchymal stromal (stem) cells (MSCs) to be mobilised from their local depot towards sites of injury and to participate in tissue repair makes these cells promising candidates for cell therapy. Physiological O2 tension in an MSC niche in vivo is about 4–7%. However, most in vitro studies of MSC functional activity are performed at 20% O2. Therefore, this study focused on the effects of short-term hypoxic stress (0.1% O2, 24 h) on adipose tissue-derived MSC motility at tissue-related O2 level. No significant changes in integrin expression were detected after short-term hypoxic stress. However, O2 deprivation provoked vimentin disassembly and actin polymerisation and increased cell stiffness. In addition, hypoxic stress induced the downregulation of ACTR3, DSTN, MACF1, MID1, MYPT1, NCK1, ROCK1, TIAM1, and WASF1 expression, the products of which are known to be involved in leading edge formation and cell translocation. These changes were accompanied by the attenuation of targeted and nontargeted migration of MSCs after short-term hypoxic exposure, as demonstrated in scratch and transwell migration assays. These results indicate that acute hypoxic stress can modulate MSC function in their native milieu, preventing their mobilisation from sites of injury.

IFN-gamma priming of adipose-derived stromal cells at “physiological” hypoxia

Multipotent mesenchymal stromal cells (MSCs) are considered cue regulators of tissue remodeling. Their activity is strongly governed by local milieu, where O2 level is most important. The elevation of inflammatory mediators and acute O2 lowering may additionally modulate MSC activity. In present paper the priming effects of IFN‐gamma on adipose tissue‐derived MSCs (ASCs) at tissue‐related O2 level (5%) and acute hypoxic stress (0.1% O2) were assessed as alterations of ASCs’ CFU‐F, proliferation, migration, osteo‐commitment. IFN‐gamma priming provoked ROS elevation, cell growth slowdown, attenuation of both spontaneous and induced osteodifferentiation of tissue O2‐adapted ASCs. The prominent changes in ASC cytoskeleton‐related gene transcription was detected. IFN‐gamma exposure shifted the ASC paracrine profile, suppressing the production of VEGF and IL‐8, while MCP‐1 and IL‐6 were stimulated. Conditioned medium of IFN‐gamma‐primed ASCs did not activate vessel growth in the CAM assay, but induced endothelial cell migration in “wound closure.” Short‐term hypoxia suppressed CFU‐F number, IFN‐gamma‐induced elevation of IL‐6 and endothelial cell migration, while it abolished IFN‐gamma‐provoked VEGF inhibition. After N‐acetyl cysteine treatment ROS level was partly abolished providing additional enhancement of IL‐6 and suppression of IL‐8 and VEGF production. These findings demonstrated that paracrine activity of ASCs in part may be governed by ROS level. Thus, this study first demonstrated that IFN‐gamma priming itself and in combination with acute O2 deprivation could supply dual effects on ASC functions providing both stimulatory and hampering effects. The equilibrium of these factors is a substantial requirement for the execution of MSC remodeling functions.

Macroporous modified poly (vinyl alcohol) hydrogels with charged groups for tissue engineering: Preparation and in vitro evaluation

Poly(vinyl alcohol) (PVA) hydrogels are widely employed for various biomedical applications, including tissue engineering, due to their biocompatibility, high water solubility, low protein adsorption, and chemical stability. However, non-charged surface of PVA-based hydrogels is not optimal for cell adhesion and spreading. Here, cross-linked macroporous hydrogels based on low molecular weight acrylated PVA (Acr-PVA) was synthesized by modification of the pendant alcohol groups on the PVA with glycidyl methacrylate (GMA). To enhance cell affinity, charged groups were introduced to the hydrogel composition. For this purpose, Acr-PVA was copolymerized with either negatively charged acrylic acid (AA) or positively charged 2-(diethylamino) ethyl methacrylate (DEAEMA) monomers. A surface charge of the obtained hydrogels was found to be in function of the co-monomer type and content. Confocal microscopy observations confirmed that adhesion and spreading of both mouse fibroblasts (L929) and human mesenchymal stem cells (hMSC) on the modified Acr-PVA-AA and Acr-PVA-DEAEMA hydrogels were better than those on the non-modified Acr-PVA hydrogel. The increase of DEAEMA monomer content from 5 to 15mol% resulted in the enhancement of cell viability which was 1.5-fold higher for Acr-PVA-DEAEMA-15 hydrogel than that of the non-modified Acr-PVA hydrogel sample.

WNT-associated gene expression in human mesenchymal stromal cells under hypoxic stress.

The effect of short-term stress on the expression of 84 Wnt-related genes in mesenchymal stromal cells (MSCs) was analyzed. In MSCs permanently expanded at 5% O2, Dkk1, RHOA, CSNK1A1, CSNK2A1, GSK3β, and PORCN genes were most highly expressed. FZD1, FZD3, SFRP1, and SFRP4 genes were up-regulated after short-term hypoxic stress (0.1% O2), whereas the expression of LEF-1 and RUVBL1 genes was significantly inhibited (down-regulated). The data suggest that Wnt signal transduction was suppressed under hypoxic stress, whereas the noncanonical Wnt signaling prevailed under the “physiological hypoxia” (5% O2).

Effects of photodynamic treatment on mesenchymal stromal cells

185 Photodynamic treatment (PDT) is a noninvasive method of cell elimination based on interaction of a photosensitizer substance and laser irradiation of low intensity [5]. This interaction leads to photosensitizer excitation, which is followed by chemical reactions yielding reactive oxygen species (ROS) that are toxic for cells. This method makes it possible to eliminate selectively individual cell populations, because various cell types differ in the ability to accumulate photosen sitizers, and their resistance to damage is also different [1, 2, 4].

Effects of Photodynamic Exposure on Endothelial Cells In Vitro

Experiments on cultured human umbilical vein endotheliocytes showed that accumulation of photosensitive dye (aluminum phthalocyanin; PHOTOSENSE) in cells and laser exposure alone were inessential for the viability of endothelial cells. Contrary to this, exposure of the cells which have accumulated aluminum phthalocyanin (an average of 111.1 ng/mg protein) to low-intensity laser (λ=675 nm) led to a dose-dependent reduction of endotheliocyte viability. Hence, cultured endothelial cells can be used for screening of various photosensitizers and preliminary optimization of photodynamic therapy.

Metal‐free Phtalocyanine and 5‐Aminolevulenic Acid in Photodynamic Treatment of Human Vascular Cells

Originally developed as a tumor therapy, now photodynamic therapy (PDT) may become a useful tool for treatment of cardiovascular diseases. Different cell types are involved in this vascular pathology, and these cells possess different susceptibility to PDT. In this study we screened the effects of two new photosensitizers (PtS and ALA) on human vascular cells. Human macrophages (Mph), aorta endothelial (HAEC) and smooth muscle (SMC) cells were obtained and cultured as described elsewhere. 2–10 ug/ml PtS was added to culture medium 24 h before PDT. ALA was added in 2–10 mM concentration in serum‐free culture medium. Then cells were washed carefully and illuminated with 692‐nm (PtS) or 633‐nm (ALA) light. Cellular viability was measured with MTT‐test. Except the case of use 5–10 mM ALA, either photosensitizer accumulation alone or laser illumination alone did not affect cells. Illumination of PtS or ALA–loaded cells (1–20 J/cm2) impaired cellular viability in dose‐dependent manner. LD90 for different vascul...

Immobilized phthalocyanines of magnesium, aluminum, and zinc in photodynamic treatment of mesenchymal stromal cells

Phthalocyanines of magnesium, aluminum, and zinc immobilized on nano-sized silica and poly-N-vinylpyrrolidone in aqueous solutions were synthesized. Photochemical activity of the immobilized metal complexes was assessed by generation of singlet oxygen. Nontoxic concentrations of the new photosensitizers were determined in vitro. A comparative analysis of the efficiency of photodynamic therapy (PDT) was performed for immobilized phthalocyanines using mesenchymal stromal cells as a cell model. Aluminum phthalocyanine immobilized on nano-sized silica displayed the highest cell tropism. Irradiation of phthalocyanine-loaded cells resulted in generation of active singlet oxygen and subsequent apoptotic cell death. The use of immobilized phthalocyanines allowed decreasing the effective concentration (dose) of photosensitizer and enhancing the PDT cytotoxicity.

Accumulation and Elimination of Photosens and Protoporphyrin IX by Different Types of Mesenchymal Cells

We studied the kinetics of accumulation and elimination of Photosens and accumulation of protoporphyrin IX in macrophages, endothelial cells, and mesenchymal stromal cells of the human adipose tissue in vitro. In all studied cells, the dynamics of Photosens accumulation was described by a multiphase curve and the maximum accumulation of the dye was observed during the second exponential phase. Elimination of Photosens did not depend on the cell function. Accumulation of protoporphyrin IX differed considerably in different cells: it was maximum in mesenchymal stromal cells was practically not detected in endothelial cells. Accumulation of the dye by macrophages depended on individual donor characteristics.