Journal: Journal of tissue engineering and regenerative medicine

Article Title: 3D matrix embedding inhibits cycloheximide-mediated sensitization to TNF-alpha-induced apoptosis of human endothelial cells

doi: 10.1002/term.2609

Figure Lengend Snippet: Human aortic ECs were cultured either on conventional 2D-polystyrene tissue culture plates (TCPS) or within 3D Gelfoam matrices (GF). A CHX-mediated sensitization to TNF-α was used to induce EC apoptosis. Results are given as the mean SD of n = 8 independent experiments. **p < 0.01.

Article Snippet: 2.1 Cell culture on 3D-matrices and on 2D-tissue culture plates Human aortic ECs were obtained from Promocell (Heidelberg, Germany) and grown in optimized endothelial growth medium-2 (Promocell) supplemented with 5% fetal bovine serum either on polystyrene-coated tissue culture plates or embedded within 3D matrices of denaturated collagen (Gelfoam; Pfizer, New York, USA) as previously published ( Methe et al., 2005 ; Nugent & Edelman, 2001 ): compressed sponges were cut into 1 × 1 × 0.3 cm blocks and hydrated in culture medium at 37 °C for ≥4 h. Then 4.5 × 10 4 ECs (suspended in ~50 μL media) were seeded onto one surface of the hydrated matrix, allowed 1.5 h to attach before turning the matrix over and seeding an additional 4.5 × 10 4 in growth media.

Techniques: Cell Culture

Journal: Journal of tissue engineering and regenerative medicine

Article Title: 3D matrix embedding inhibits cycloheximide-mediated sensitization to TNF-alpha-induced apoptosis of human endothelial cells

doi: 10.1002/term.2609

Figure Lengend Snippet: ECs were either cultured on conventional 2D-polystyrene tissue culture plates with (TCPS) or without (CRTL) addition of TNF-α and CHX or within a Gelfoam matrix with addition of TNF-α and CHX. Results show the mean ± SD of n = 8 independent Western blots without induction of EC-apoptosis (Figure 3C), after 15 minutes (Figure 3A) and 1 hour (Figure 3B) of stimulation TNF-α and CHX. *p < 0.05; **p < 0.01.

Article Snippet: 2.1 Cell culture on 3D-matrices and on 2D-tissue culture plates Human aortic ECs were obtained from Promocell (Heidelberg, Germany) and grown in optimized endothelial growth medium-2 (Promocell) supplemented with 5% fetal bovine serum either on polystyrene-coated tissue culture plates or embedded within 3D matrices of denaturated collagen (Gelfoam; Pfizer, New York, USA) as previously published ( Methe et al., 2005 ; Nugent & Edelman, 2001 ): compressed sponges were cut into 1 × 1 × 0.3 cm blocks and hydrated in culture medium at 37 °C for ≥4 h. Then 4.5 × 10 4 ECs (suspended in ~50 μL media) were seeded onto one surface of the hydrated matrix, allowed 1.5 h to attach before turning the matrix over and seeding an additional 4.5 × 10 4 in growth media.

Techniques: Cell Culture, Western Blot

Journal: Journal of tissue engineering and regenerative medicine

Article Title: 3D matrix embedding inhibits cycloheximide-mediated sensitization to TNF-alpha-induced apoptosis of human endothelial cells

doi: 10.1002/term.2609

Figure Lengend Snippet: Figure 6A shows phosphorylation of p38-MAPK at Thr180/Tyr182 after treatment with a combination of TNF-α and CHX for 10 minutes for ECs on 2D-TCPS and in a Gelfoam matrix (GF). An unstimulated 2D-control culture (CRTL) without addition of cytokines was evaluated. Figure 6B shows basal protein expression of p38-MAPK as determined by western blot for culture of ECs on conventional 2D-polystyrene tissue culture plates (TCPS) and in a 3D Gelfoam matrix (GF). For evaluation of basal expression no cytokines were added. Results show the mean ± SD of n = 8 independent western blots. *p < 0.05; **p < 0.01; ns = not significant.

Article Snippet: 2.1 Cell culture on 3D-matrices and on 2D-tissue culture plates Human aortic ECs were obtained from Promocell (Heidelberg, Germany) and grown in optimized endothelial growth medium-2 (Promocell) supplemented with 5% fetal bovine serum either on polystyrene-coated tissue culture plates or embedded within 3D matrices of denaturated collagen (Gelfoam; Pfizer, New York, USA) as previously published ( Methe et al., 2005 ; Nugent & Edelman, 2001 ): compressed sponges were cut into 1 × 1 × 0.3 cm blocks and hydrated in culture medium at 37 °C for ≥4 h. Then 4.5 × 10 4 ECs (suspended in ~50 μL media) were seeded onto one surface of the hydrated matrix, allowed 1.5 h to attach before turning the matrix over and seeding an additional 4.5 × 10 4 in growth media.

Techniques: Expressing, Western Blot

Journal: Journal of tissue engineering and regenerative medicine

Article Title: 3D matrix embedding inhibits cycloheximide-mediated sensitization to TNF-alpha-induced apoptosis of human endothelial cells

doi: 10.1002/term.2609

Figure Lengend Snippet: Figure 8A shows phosphorylation of FAK at Tyr397 as determined by Western Blot (n=8). ECs cultured on conventional 2D-tissue culture plates (TCPS) or in a 3D Gelfoam matrix (GF) were stimulated with a combination of TNF-α and CHX for 10 minutes. An unstimulated culture of ECs on conventional 2D-polystyrene culture plates served as control (CRTL). Figure 8B shows basal protein expression of FAK as determined by Western blot, comparing culture of ECs on 2D-TCPS and in a Gelfoam matrix (GF). When comparing basal protein expression no cytokines were added. Results show the mean ± SD of n = 8 independent western blots. *p < 0.05; **p < 0.01; ns = not significant.

Article Snippet: 2.1 Cell culture on 3D-matrices and on 2D-tissue culture plates Human aortic ECs were obtained from Promocell (Heidelberg, Germany) and grown in optimized endothelial growth medium-2 (Promocell) supplemented with 5% fetal bovine serum either on polystyrene-coated tissue culture plates or embedded within 3D matrices of denaturated collagen (Gelfoam; Pfizer, New York, USA) as previously published ( Methe et al., 2005 ; Nugent & Edelman, 2001 ): compressed sponges were cut into 1 × 1 × 0.3 cm blocks and hydrated in culture medium at 37 °C for ≥4 h. Then 4.5 × 10 4 ECs (suspended in ~50 μL media) were seeded onto one surface of the hydrated matrix, allowed 1.5 h to attach before turning the matrix over and seeding an additional 4.5 × 10 4 in growth media.

Techniques: Western Blot, Cell Culture, Expressing

Journal: Journal of tissue engineering and regenerative medicine

Article Title: 3D matrix embedding inhibits cycloheximide-mediated sensitization to TNF-alpha-induced apoptosis of human endothelial cells

doi: 10.1002/term.2609

Figure Lengend Snippet: Association of RIP with FAK were analyzed by Western Blot using anti-RIP-IgG-antibodies was compared in ECs cultured on TCPS with ECs cultured within a 3D Gelfoam matrix (GF) after addition of TNF-α and CHX. Additionally, both conditions were compared to an unstimulated control 2D-culture on TCPS (CRTL) without addition of cytokines. Results show the mean ± SD of n = 8 independent western blots. **p < 0.01; ns = not significant.

Article Snippet: 2.1 Cell culture on 3D-matrices and on 2D-tissue culture plates Human aortic ECs were obtained from Promocell (Heidelberg, Germany) and grown in optimized endothelial growth medium-2 (Promocell) supplemented with 5% fetal bovine serum either on polystyrene-coated tissue culture plates or embedded within 3D matrices of denaturated collagen (Gelfoam; Pfizer, New York, USA) as previously published ( Methe et al., 2005 ; Nugent & Edelman, 2001 ): compressed sponges were cut into 1 × 1 × 0.3 cm blocks and hydrated in culture medium at 37 °C for ≥4 h. Then 4.5 × 10 4 ECs (suspended in ~50 μL media) were seeded onto one surface of the hydrated matrix, allowed 1.5 h to attach before turning the matrix over and seeding an additional 4.5 × 10 4 in growth media.

Techniques: Western Blot, Cell Culture