EBMTM Endothelial Cell Growth Basal Medium, 500 mL

Protocol tips

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	To reduce the ‘edge effect’ in a 96-well plate: fill the outer edge wells with 200 μL D-PBS or BBB working medium, use only the inner wells of the plate for culturing the BBB organoids

Protocol tips
To reduce the ‘edge effect’ in a 96-well plate: fill the outer edge wells with 200 μL D-PBS or BBB working medium, use only the inner wells of the plate for culturing the BBB organoids

Publication protocol

Organoid culture – TIMING 48 hrs: Calculate the volume needed to seed 1.5 × 103 cells of hCMEC/D3 (or HBMEC), HBVP and astrocytes. To achieve a volume of 200 μL per well in a 96-well agarose plate, subtract the total cell volume from 200 μL, and pipette that amount of BBB working medium into each well using a multi-channel pipette. Then, add the appropriate volume of each cell type using a multi-channel pipette into each well containing the BBB working medium. The final volume in each well should be 200 μL. Δ CRITICAL STEP To ensure proper cell seeding, assess each well under an inverted light microscope after each seeding step. To reduce the ‘edge effect’ in a 96-well plate, where the level of evaporation at the edge wells is much higher, fill the outer edge wells with 200 μL of either D-PBS or BBB working medium to act as a moisture barrier, and use only the inner wells of the plate for culturing the BBB organoids. Incubate the plate at 37°C in a humidified CO2 incubator for 48h. The organoids should assemble properly into compact spheroids within 12–24 hrs (Fig. 2a). After the 48 hrs, assess the organoid quality in each well prior to collecting them for experimental purposes (see Fig. 2b as a general guideline). Proceed to the next steps only with organoids that have assembled properly. Collect each good organoid by pipetting, and pool them together in a microcentrifuge tube for experimental use (Fig. 2c). CRITICAL STEP Organoid quality can be further assessed by immuno-staining example organoids for expression of markers of the BBB, such as tight junctions (ZO-1) and efflux pump (P-glycoprotein) as illustrated in Fig. 2d and andee. BBB working medium. For organoid formation using hCMEC/D3, add endothelial growth supplements hEGF, hydrocortisone, GA-100, hFGF-B, R3-IGF-1, ascorbic acid and heparin to 500 mL of endothelial basal medium (EBM from Lonza) in a sterile tissue culture hood. Supplement this medium with 2% human serum. Invert bottle a few times to ensure thorough mixing of all components. Store bottle at 2–8°C in the fridge for no longer than 6 weeks.! Critical Do not add FBS (since medium has already been supplemented with human serum) and VEGF (as VEGF will disrupt the tight junctions on the organoid surface) to this medium. ! Critical For organoid formation using primary HBMEC, add 2% human serum to endothelial basal medium (from ScienCell) and exclude supplementation with FBS and ECGS.

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