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Figure 4 | Fluids and Barriers of the CNS

Figure 4

From: Exploring the effects of cell seeding density on the differentiation of human pluripotent stem cells to brain microvascular endothelial cells

Figure 4

Day 0 hPSC starting density can be used to scale down brain microvascular endothelial cell differentiation from traditional 6-well culture to 12-, 24-, 48-, and 96-well cultures. a i Brightfield images of IMR90-4-derived BMECs differentiated in 6-, 12-, 24-, 48-, or 96-well format for 8 days. Scale bars 1 mm. ii Immunostaining of purified IMR90-4-derived BMECs at day 10 following differentiation in either 6-, 12-, 24-, 48-, or 96-well plate format. Scale bars 50 μm. b i Maximum TEER of IMR90-4-derived BMECs differentiated in either 6-, 12-, or 24-well format and purified by subculturing onto 12-well Transwells at a filter seeding density of 1 million cells/cm2. Data from each 6-, 12-, and 24-well differentiation were paired and averaged from four independent differentiations. ii Maximum TEER of IMR90-4-derived BMECs differentiated in 6-well format and subcultured onto either 12- or 24-well Transwells at a filter seeding density of 1 million cells/cm2. Data from 12- and 24-well Transwells were paired and averaged from two independent differentiations. c IMR90-4-derived BMECs were differentiated in 6-, 12-, 24-, 48-, or 96-well format, and efflux transporter inhibition of purified cultures at day 10 was measured via intracellular accumulation of i rhodamine 123 or ii DCFDA. Inhibitor-treated samples were independently normalized to each respective non-inhibitor-treated control sample. Statistical significance was calculated using Student’s unpaired t test (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). Values are mean ± SD of three replicates from a single differentiation, and experiments were repeated for two additional independent differentiations for verification of reported trends. All differentiations in panels ac were initiated at a day 0 hPSC starting density of 30,000 cells/cm2.

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