Spheroids were generated as previously reported [25 (link)]. Briefly, cells resuspended in endothelial basal medium (EBM) containing 0.24% high viscosity methylcellulose (Sigma-Aldrich), and were seeded in 96-well round bottom non-adherent plates. Cells were incubated overnight at 37 °C. Each spheroid contained 1 × 103 cells. Individual spheroids were harvested, embedded in rat tail collagen type 1 gel (Corning, Seneffe, Belgium), supplemented with EBM, and incubated for 48 h at 37 °C. To quantify germination, the average number of shoots under each condition was calculated.
Rat tail collagen type 1 gel
Manufactured by Corning
Sourced in Belgium
Rat tail collagen type 1 gel is a laboratory product derived from the collagen found in rat tails. It is a viscous solution that can be used as a substrate for cell culture and tissue engineering applications. The gel provides a natural extracellular matrix-like environment for cells to grow and proliferate.
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Lab products found in correlation
4 protocols using rat tail collagen type 1 gel
1
Spheroid-based Angiogenesis Assay
2
Sprouting Angiogenesis Assay Using Spheroids
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To generate the spheroids, we proceeded as previously reported [41 (link)]. Briefly, HUVECs resuspended in EBM containing 0.24% high viscosity methyl cellulose (Sigma-Aldrich) were seeded in 96 well round bottom non-adherent plates and cultured overnight at 37°C. Each spheroid contained 103 cells. Single spheroids were collected, embedded in rat tail collagen type 1 gel (Corning, Seneffe, Belgium) and cultured for 48 hours at 37°C in 2% FBS supplemented EBM with 75 ng/ml phorbol-12 myristate 13-acetate (PMA) or 10 ng/ml b-FGF. To quantify the sprouting, the mean number of sprout in each condition was counted.
3
Reconstructed Human Skin Protocol
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Primary cells were from Lonza (Normal Human Neonatal Dermal Fibroblasts – Catalog #: CC-2509 and Human Neonatal Epidermal Keratinocytes, pooled – Catalog #: 00192906). The reconstructed skin was prepared in 2 steps. Briefly, the dermal compartment was prepared using rat tail type I collagen gel (Corning) and 1.5 × 105 primary human dermal fibroblasts per construct. After polymerizing the collagen gel for 2 hours, 2.5 × 105 primary human epidermal keratinocytes were seeded on top of each construct and kept submerged in RAFT: KGM-Gold Bullet Kit Medium (1:1) for 24 hours. The inserts were then raised and maintained at the air–liquid interface for 10 days for stratification of the skin. The 3D skin was maintained in a 5% carbon dioxide incubator with low humidity (∼50%).
4
Constructing Pigmented Skin Model
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The skin was prepared in two steps as previously described [14 (link)]. Briefly, the dermal compartment was prepared using rat tail type-I collagen gel (Corning, Tewksbury, MA) and 1.5 × 105 Fibroblasts per construct. After polymerization of the collagen gel for 2 h, 2.5 × 105 keratinocytes and 1.7 × 105 melanocytes were seeded on top of each construct and were kept submerged in RAFT: KGM-Gold Bullet Kit Medium (1:1) for 24 h. The inserts were then raised and maintained at the air-liquid interface for 12 d for the stratification of the skin. The 3D skin was maintained in a 5% CO2 incubator with low humidity (~50%), and the medium supplemented with tyrosine (0.25 mM) + NH4Cl (5 mM) was replaced every 2 d.
Pigmented or unpigmented skin was fixed in 10% buffered formalin at 4°C for 12 h, followed by dehydration and cleaning in solutions containing increasing concentrations of alcohol and xylene for paraffin inclusion. Paraffin sections (5 μm) were stained with hematoxylin and eosin (H&E) for morphological analysis and Fontana-Masson for melanin content analysis. All images were obtained by optical microscopy (Olympus Life Science-40×) and analyzed using NIS-Elements software (Nikon Instruments, Melville, NY).
Pigmented or unpigmented skin was fixed in 10% buffered formalin at 4°C for 12 h, followed by dehydration and cleaning in solutions containing increasing concentrations of alcohol and xylene for paraffin inclusion. Paraffin sections (5 μm) were stained with hematoxylin and eosin (H&E) for morphological analysis and Fontana-Masson for melanin content analysis. All images were obtained by optical microscopy (Olympus Life Science-40×) and analyzed using NIS-Elements software (Nikon Instruments, Melville, NY).
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