EAhy 926 attached to GEM™.

<p>Nuclear staining with 5 µg/ml Hoechst 33342 was performed 1, 5, 7, and 14 days after inoculation (A). Vital dye staining for ER and mitochondria five days after inoculation. Hoechst 33342 dye was used as nuclear counterstain (B). Internalized red fluorescent NPs co-localize with the lysosomal dye LysoSensor™ Green DND-189 but not with the nucleus (blue) (C).</p

Mode of action of PPS in conventional cultures.

<p>Activation of caspases 3 and 7 (A and B) and release of LDH (C and D) upon exposure of EAhy 926 cells to 20 and 200 nm PPS for 4, 8, and 24 hours compared to untreated cells. Data are presented as mean ± SD. (h), hours.</p

Growth curve of EAhy 926 cultured on basal membrane coated GEM™.

<p>Two pre-installed protocols for cell culturing epithelial (HEK 293) and endothelial (HUVEC) cells were compared. (d), days.</p

Acute cytotoxicity of NPs exerted on EAhy 926 in different cell cultures after 24 hours.

<p>Cells in conventional cultures were treated with NPs dissolved in serum-free medium (A) as well as in medium with 10% FBS (B). Cells cultured on microcarriers were exposed to NMs dissolved in medium with 10% FBS. Data are presented as mean ± SD.</p

Development of an Advanced Intestinal in Vitro Triple Culture Permeability Model To Study Transport of Nanoparticles

Intestinal epithelial cell culture models, such as Caco-2 cells, are commonly used to assess absorption of drug molecules and transcytosis of nanoparticles across the intestinal mucosa. However, it is known that mucus strongly impacts nanoparticle mobility and that specialized M cells are involved in particulate uptake. Thus, to get a clear understanding of how nanoparticles interact with the intestinal mucosa, in vitro models are necessary that integrate the main cell types. This work aimed at developing an alternative in vitro permeability model based on a triple culture: Caco-2 cells, mucus-secreting goblet cells and M cells. Therefore, Caco-2 cells and mucus-secreting goblet cells were cocultured on Transwells and Raji B cells were added to stimulate differentiation of M cells. The in vitro triple culture model was characterized regarding confluence, integrity, differentiation/expression of M cells and cell surface architecture. Permeability of model drugs and of 50 and 200 nm polystyrene nanoparticles was studied. Data from the in vitro model were compared with ex vivo permeability results (Ussing chambers and porcine intestine) and correlated well. Nanoparticle uptake was size-dependent and strongly impacted by the mucus layer. Moreover, nanoparticle permeability studies clearly demonstrated that particles were capable of penetrating the intestinal barrier mainly via specialized M cells. It can be concluded that goblet cells and M cells strongly impact nanoparticle uptake in the intestine and should thus be integrated in an in vitro permeability model. The presented model will be an efficient tool to study intestinal transcellular uptake of particulate systems

Analysis of the cytotoxic effect of chemotherapeutic agents on ALDH1^high and ALDH1^low populations sorted from (A–C) SW-982 and (D–F) SW-1353 cells.

<p>Both subopulations were treated with 0–5.0 µM doxorubicin, 0–5.0 µM epirubicin, 1–100 µM cisplatin, and measured after 48 h. Mean value ±SD of all measurements was fitted according the Hill equation. Significant differences on the individual concentrations were incorporated in the curves.</p

Primer Sequences used for real-time RT-PCR.

<p>Primer Sequences used for real-time RT-PCR.</p

Proliferation analysis of ALDH1^high and ALDH1^low sarcoma cells.

<p>The immunohistochemical analysis using anti-Ki-67 proliferation marker revealed a decreased proliferation level of (A) SW-1353 ALDH1^low cells and compared to (B) SW-1353 ALDH1^high cells. (C–E) Dynamic proliferation curves for ALDH1^high and ALDH1^low cells seeded at 10,000 cells per well measured with the xCELLigence system.</p

Overview of all results including the corresponding significances.

<p>Overview of all results including the corresponding significances.</p

Aldehyde dehydrogenase 1 (ALDH1) expression in sarcoma cell lines using the Aldefluor® assay.

<p>Fluorescence versus forward scatter was shown in a density blot from (A) DEAB control cells and (B) ALDH1-expressing cells (called ALDH1^high). (C) ALDH1 expression in % of gated cells. The highest proportion of ALDH1^high cells is represented by SW-684 cells (1.77±0.9%; n = 12), SW-982 cells (2.23±1.0%; n = 11), and SW-1353 cells (2.69±1.3%; n = 8). (D) After two weeks cultured the ALDH1^high population generated a significant higher account of ALDH1^high cells. (E) The enhanced ALDH activity was also demonstrated by western blot.</p