Assay protocols were adapted from those developed for primary endothelial cells [7 (link)]. 150 μm diameter Cytodex™ 3 microcarrier beads (GE Healthcare) were prepared according to manufacturer recommendations. Beads were coated overnight with 50 μg mL−1 human placental collagen IV (Sigma) and 25 μg mL−1 fibronectin from human plasma (Sigma). dhBMECs were singularized using 30 minute StemPro accutase (ThermoFisher) treatment and incubated at a ratio of 1000:1 (dhBMECs:beads) for two hours under gentle agitation every 30 minutes. “Bead seeding media” was comprised of human endothelial cell serum-free media (Life Technologies) supplemented with 1% human platelet poor derived serum (Sigma), 1% Penicillin Streptomycin (Thermo Fisher), 2 ng mL−1 bFGF (R&D Systems), 10 μM all-trans retinoic acid (Sigma), and 10 μM ROCK inhibitor Y27632 (RI). Inclusion of RI was required to enable cell adhesion, as previously found for collagen-based biomaterials [32 (link)]. After two hours, non-adherent dhBMECs were removed and beads were cultured for 24 hours on a shaker at 100 rpm in bead seeding media. To form HUVEC coated beads, identical protocols were used with the following differences: (1) incubation with cells for only one hour, (2) use of HUVEC media without supplementation with RI.
Human placental collagen 4
Manufactured by Merck Group
Human placental collagen IV is a purified, biologically-derived extracellular matrix protein. It serves as a structural component in cell culture applications.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Stempro accutase, by Thermo Fisher Scientific (2 mentions)
Basic fibroblast growth factor (bfgf), by R&D Systems (2 mentions)
Cytodex 3 microcarrier beads, by GE Healthcare (2 mentions)
Fibronectin from human plasma, by Merck Group (2 mentions)
Human platelet poor derived serum, by Merck Group (2 mentions)
Human endothelial cell serum free media, by Thermo Fisher Scientific (2 mentions)
Transwell insert, by Corning (1 mentions)
Nhbe cells, by Lonza (1 mentions)
Dmem ham s f12, by Thermo Fisher Scientific (1 mentions)
Insulin, by Merck Group (1 mentions)
Isoproterenol, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Lonza (1 mentions)
Penicillin streptomycin p s, by Thermo Fisher Scientific (1 mentions)
0.4 μm pore polyester membrane insert, by Corning (1 mentions)
Endohm, by World Precision Instruments (1 mentions)
Rat tail collagen 1, by Merck Group (1 mentions)
6 protocols using human placental collagen 4
1
Endothelial Cell Coating of Microcarrier Beads
2
Differentiation of NHBE Cells
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Cryopreserved primary normal human bronchial epithelial (NHBE) cells (Lonza) were seeded at a density of 1.5 × 105 cells/cm2 on human placental collagen IV (Sigma-Aldrich) coated Transwell® inserts (Corning 3470). Cells were cultured in lung medium77 (link) until confluence (2–3 days), and then maintained at the air-liquid interface with basal media changes every 2 days for three weeks to induce differentiation into ciliated and secretory cell populations.
3
Keratinocyte and Fibroblast Co-culture
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Keratinocytes and fibroblasts were isolated from skin and gingiva tissue and cultured as described earlier [28 (link)]. Fibroblasts (passage 3) were seeded as a monoculture at a density of 7 × 103 cells/cm2 in 6-well culture plates in fibroblast medium, consisting of Dulbecco's modified Eagle medium (DMEM) (Lonza, Verviers, Belgium) containing 1% Ultroser G (UG) (BioSepra, Cergy-Saint-Christophe, France) and 1% penicillin-streptomycin (P/S) (Gibco). Keratinocytes (passage 2) were seeded as a monoculture at a density of 4 × 104 cells/cm2 in keratinocyte medium in 6-well culture plates, precoated with 0.5 μg/cm2 human placental collagen IV (Sigma-Aldrich). Keratinocyte medium consisted of DMEM/Ham's F-12 (Gibco) (3 : 1), 1% UG, 1% P/S, 1 μM isoproterenol (Sigma-Aldrich), and 0.1 μM insulin (Sigma-Aldrich). For cocultures, first, the fibroblasts were seeded at a density of 7 × 103 cells/cm2 on collagen IV-coated 6-well plates in fibroblast medium. After initial attachment of fibroblasts (4 h), the keratinocytes were seeded in the same 6-well plates at a density of 2.4 × 103 cells/cm2 in keratinocyte medium. This results in a well with 75% fibroblasts and 25% keratinocytes. After initial attachment of keratinocytes (4 h), the medium was switched to keratinocyte medium.
4
Characterizing Blood-Brain Barrier Permeability
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iBMECs were seeded at a density of 1 × 106 cm−2 onto Transwells that were treated overnight with 50 μg mL−1 human placental collagen IV (Sigma) and 25 μg mL−1 fibronectin from human plasma (Sigma). Transendothelial electrical resistance (TEER) values (Ω cm2) were recorded using an EndOhm (World Precision Instruments), as previously reported [23 (link)]. All measurements were performed on 6.5 mm Transwells with a 0.4 μm pore polyester membrane insert (Corning). TEER values for Transwells with no cells were subtracted from the measured values, and were then normalized to the membrane area. The effects of antibodies, cancer-conditioned medium, and co-cultured cancer cells on TEER were tested (Additional file 1 : Fig. S5). The apical chambers of Transwells were exposed to identical concentrations of the fluorescently-labeled molecules as used in 3D assays for 24 h. Conditioned medium was collected from microvessels as the downstream perfusate and stored at −80 °C. The direct effects of co-cultured cancer cells on TEER were tested by seeding the basolateral chamber of Transwells with ~ 50 cancer spheroids, matching the ratio of iBMECs to spheroids used in 3D models.
5
Cell Adhesion Assay Protocol
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Cell adhesion assays were performed in 96-well plates as previously described (Chen et al., 2004 (link)). Cells (1 × 105) were seeded in serum-free medium onto plates containing different concentrations of ECM for 60 min. Adherent cells were fixed, stained with crystal violet, and solubilized, and the optical densities of the cell lysates were read at 570 nm (OD570). Human placental collagen IV and rat tail collagen I were purchased from Sigma-Aldrich, St Louis, MO.
6
Endothelial Cell Culture on Microcarrier Beads
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Assay protocols were adapted from those developed for primary endothelial cells [6] . 150 µm diameter Cytodex™ 3 microcarrier beads (GE Healthcare) were prepared according to manufacturer recommendations. Beads were coated overnight with 50 µg mL - 1 human placental collagen IV (Sigma) and 25 µg mL - 1 bronectin from human plasma (Sigma). dhBMECs were singularized using 30 minute StemPro accutase (ThermoFisher) treatment and incubated at a ratio of 1000:1 (dhBMECs:beads) for two hours under gentle agitation every 30 minutes. "Bead seeding media" was comprised of human endothelial cell serum-free media (Life Technologies) supplemented with 1% human platelet poor derived serum (Sigma), 1% Penicillin Streptomycin (Thermo Fisher), 2 ng mL - 1 bFGF (R&D Systems), 10 µM alltrans retinoic acid (Sigma), and 10 µM ROCK inhibitor Y27632 (RI). Inclusion of RI was required to enable cell adhesion, as previously found for collagen-based biomaterials [29] . After two hours, non-adherent dhBMECs were removed and beads were cultured for 24 hours on a shaker at 100 rpm in bead seeding media. To form HUVEC coated beads, identical protocols were used with the following differences: (1) incubation with cells for only one hour, (2) use of HUVEC media without supplementation with RI.
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