![]() There are many black particles when cultured in osteogenic media (right). ( B) Representative high-resolution SE-ADM images of osteoblasts cultured with or without osteogenic media for 7 days. Black particles were evident only when cultured in osteogenic media (right, square in the bottom). ( A) Representative high-resolution SE-ADM images of osteoblasts cultured with or without osteogenic media for 2 days. However, we cannot rule out the possibility that these particles precipitated spontaneously in the extracellular space after secretion from the cell. The particles observed in osteogenic media ranged from 4 to 320 nm, with a median size of 90 nm, which was consistent with the previously reported size of MVs ( 6, 8), suggesting that they are indeed MVs. When we measured the size of 900 to 1100 particles on the image for each and calculated the diameter, the average particle size was 98 nm on day 4 and gradually increased to 117 nm on day 7 and 223 nm on day 10 ( Fig. 1E), suggesting fusion or particle growth. Secreted particles increased their size during the osteogenic culture ( Fig. To analyze the size of these secreted particles, we used SE-ADM after 4 to 10 days of culture in osteogenic media ( Fig. 1C, right) with some tendency to align to collagen fibrils (fig. Observation of MVs at later stages revealed that MVs were ubiquitously distributed ( Fig. 1C, right), which is concurrent with previous reports that the secreted MVs are located in the narrow gap between collagen fibrils ( 18). High-resolution SE-ADM observation of the remaining film revealed that most particles were firmly attached to the film ( Fig. To determine whether they were localized in the intracellular or extracellular space, we manually detached the cell monolayer from the film, using forceps, and observed the film again. At a later stage (after 7 days culture), numerous particles were visible when cultured in osteogenic media, although not in normal media ( Fig. The particles seemed to be contained in vesicles ( Fig. 1A, right), but not in normal media ( Fig. At an early stage of differentiation (day 2), black particles were present when cultured in osteogenic media ( Fig. The cells in the confluent monolayer were further cultured in normal or osteogenic media containing ascorbic acid and phosphate. S1A), and normal intracellular structures were identified ( 15). Thereafter, high-resolution SE-ADM was performed to observe cells in culture (fig. S1D) in the SE-ADM system, proliferated, and differentiated to form calcified nodules on the film (fig. Using an optical phase-contrast microscope, we first confirmed that cells in culture successfully formed a monolayer attached to the thin SiN film (fig. When the cells were cultured with ascorbic acid and phosphate for 3 days, calcified nodules formed as revealed through Alizarin Red S staining (fig. The cells stably expressed transcription factors necessary for osteoblast differentiation (fig. As an osteoblast cell line, we used the murine osteoblastic cell line KUSA-A1, which has a high osteogenic capacity in vitro and in vivo ( 17). The system setup for observing osteoblastic MVs is illustrated in fig. Here, we explored the possibility of visualizing MVs in intact osteoblasts with high-resolution SE-ADM with the aim of clarifying the biogenesis and trafficking of MVs. To overcome these limitations, we used a novel microscopic system, scanning electron-assisted dielectric microscopy (SE-ADM), wherein both nanoscale resolution and high-contrast imaging were achieved for mammalian cells in aqueous media without staining ( 15, 16).
0 Comments
Leave a Reply. |
Details
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |