Storage stability and delivery potential of cytochalasin B induced membrane vesicles

Cell-free therapies based on extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) are considered as a promising tool for stimulating regeneration and immunomodulation. The need to develop a practical approach for large-scale production of vesicles with homogenous content led to the implementation of cytochalasin B-induced to induce microvesicles (CIMVs) biogenesis. CIMVs mimic natural EVs in size and composition of the surrounding cytoplasmic membrane. Previously we observed that MSC derived CIMVs demonstrate the same therapeutic angiogenic and immunomodulatory activity as the parental MSCs, making them a potentially scalabale cell-free therapeutic option. However, little is known about their storage stability and delivery potential. Therefore, in this study, we determined the effects of different storage conditions (+37°C in serum, +4°C,-20°, +25°C in saline, as well as freeze-drying prior to storage at-20°C) on the integrity and effective delivery of CIMVs derived from human MSCs. We determined that different storage conditions alter the protein concentration within the solution used to store CIMVs over time, this concided with a decrease in the amount of CIMVs due to gradual degradation. We established that freezing and storage CIMVs in saline at-20°C reduces degredation and prolongs their shelf life. Additionally, we found that freeze-thawing preserved the CIMVs morphology better than freeze drying and subsequent rehydration which resulted in aggregation of CIMVs. Collectively our data demonstrates for the first time, that the most optimal method of CIMVs storage is freezing at-20°C, to preserve the CIMVs in the maximum quantity and quality with retention of effective delivery. These findings will benefit the formation of standardized protocols for the use of CIMVs for both basic research and clinical application

ОСОБЕННОСТИ ФОРМИРОВАНИЯ ТОНКИХ ПЛЕНОК КРЕМНИЯ, ОСАЖДАЕМЫХ МАГНЕТРОННЫМ РАСПЫЛЕНИЕМ

The surface morphology and optical properties of Si coatings formed by magnetron sputtering were studied using atomic force microscopy, scanning electron microscopy, and spectrophotometry methods. The possibility to influence the surface morphology of coating (filamentous structures and/or round holes) and the location of maxima and minima in reflectance and transmittance via a controllable variation of magnetron sputtering regimes (substrate temperature and bias potential) is shown. Методами атомно-силовой и сканирующей электронной микроскопии, а также спектрофотометрии исследованы морфология поверхности и оптические характеристики тонких Si-покрытий, сформированных методом магнетронного распыления. Показано, что при контролируемой вариации технологических параметров магнетронного распыления таких, как температура подложки и потенциал смещения, можно менять морфологию поверхности пленок Si. Для некоторых режимов осаждения обнаружено появление на поверхности нитевидных структур и/или круглых углублений, изменения положения минимумов и максимумов в оптических спектрах отражения и пропускания.

Effect of Melanization on Thallus Microstructure in the Lichen Lobaria pulmonaria

Lichens often grow in microhabitats where they experience severe abiotic stresses. Some species respond to high UV radiation by synthesizing dark brown melanic pigments in the upper cortex. However, unlike the melanized structures of non-lichenized fungi, the morphology of the melanic layer in lichens remains unstudied. Here, we analyzed the morphology, ultrastructure, and elemental composition of the melanized layer in UV-exposed thalli of the lichen Lobaria pulmonaria (L.) Hoffm. Using light microscopy, we detected a pigmented layer sensitive to staining with 3,4-L-dihydroxyphenylalanine, a precursor of eumelanin, in the upper cortex of melanized thalli. Analysis of cross-sections of melanized thalli using scanning electron microscopy revealed that melanin-like granules are deposited into the hyphal lumens. Melanized thalli also possessed thicker hyphal cell walls compared to pale thalli. Energy-dispersive X-ray spectroscopy analysis of the elemental composition of the hyphal walls and extracted melanin indicated that the type of melanin synthesized by L. pulmonaria is eumelanin. Transmission electron microscopy was used to show that during melanization melanosome-like dark vesicles are transported to the cell surface and secreted into the cell walls of the fungal hyphae. Results from this study provide new insights into the effects of melanin synthesis on the microstructure of lichen thalli