Transmission electron microscopy reveals ultrastructural details of cells, and it is a valuable method for studying cell organelles. That is why we used this method for detailed morphological description of different adult tissue-derived stem cells, focusing on the morphological signs of their functions (proteosynthetic activity, exchange with external environment, etc.) and their comparison. Preparing a specimen from the cell culture suitable for transmission electron microscopy is, however, much more challenging than routine tissue processing for normal histological examination. There are several issues that need to be solved while working with cell pellets instead of solid tissue. Here we describe a simple protocol for the isolation and culture of mesenchymal stem cells from different adult tissues, with applications to stem cell biology and regenerative medicine. Since we are working with population of cells that was obtained after many days of passaging, very efficient and gentle procedures are highly necessary. We demonstrated that our semi-conservative approach regarding to histological techniques and processing of cells for transmission electron microscopy is a well reproducible procedure which results in quality pictures and images of cell populations with minimum distortions and artifacts. We also commented about riskiest steps and histochemical issues (e.g., precise pH, temperature) while preparing the specimen. We bring full and detailed procedures of fixation, post-fixation, infiltration, embedding, polymerization and contrasting of cell obtained from in vitro cell and tissue cultures, with modifications according to our experience. All this steps are essential for us to know more about adult stem cells derived from different sources or about other random cell populations. The knowledge about detailed ultra-structure of adult stem cells cultured in vitro are also essential for their using in regenerative medicine and tissue engineering.
Background: Trichomonas vaginalis infectious disease is an important worldwide health problem. Although, several drugs especially metronidazole and tinidazole have been used in the treatment, their-resistant strains have been developed and unpleasant adverse effects exist. Garlic and other Allium species are old medicinal plants, which were used for infectious diseases, cardiovascular disorders and hair and skin cosmetic diseases. Objectives: We aimed to compare the anti-trichomoniasis activities of three species of Allium. Methods: The Trichomonas vaginalis strain was isolated from vaginal discharge of women with vaginitis symptoms and cultured in a modified TYM medium. After confirmation of herbarium of the plants, their extracts were prepared. For testing, 24 sterile plates were used. In all 24 homes, 200 µL of TYM were poured out. The tube containing the parasite, counting the number of angels with the aid of Neubauer slide and set to 500,000 per mL, was added to the amount of 100 µL to all wells. In order to investigate the antitrichomoniasis of the plant, concentrations of 37.5, 75, 150, 300, 600, 1200 and 2400 µg/mL of extract have been tested. For positive control, 50 mL of metronidazole, 50 µg/mL, was added to culture medium. In addition to, no herbal extract and metronidazole was added to the well of negative control. Results: The leaf extract of bell circus with a concentration of 2000 µg/mL after 48 hours and boiled cabbage bulb onion at a concentration of 24 µg/mL and leaf extract of turmericum at a concentration of 1500 µg/mL in 48 hours and boiled horseradish onions at 48 µg/mL concentration in 24 hours could achieve 100% GI and significantly inhibit parasite growth. Leaf extract, boiled onions and essential oils inhibited the growth of Trichomonas parasites, were not as high as 100%. Conclusions: According to the results obtained in our study, these Allium plants have inhibitory activity on growth of Trichomonas vaginalis parasite.
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