culture inserts consisting of a transparent polyethylene terephthalate (pet) membrane  (Greiner Bio)

Journal: bioRxiv

Article Title: SSAO-mediated decrease of endothelial BDNF release affects neuronal GluA1 and PSD95 expression: a peripheral mechanism inducing NVU and CNS disturbances

doi: 10.1101/2025.03.04.641410

Figure Lengend Snippet: Neurovascular unit (NVU) in vitro model A, Extended experimental timeline. Upper arrow represents neuron-glia mixed culture timeline, indicated as days in vitro (DIV). Medium changes needed at each time point are indicated. Middle arrow represents hCMEC/D3 endothelial cell line culture timeline. Seeding, confluence, and deprivation steps are indicated and were synchronized to mixed culture development. Day 0 is considered the day when cells achieve confluence and after this, time of this culture is indicated as days after confluence (DAC). Lower arrow represents the timeline once co-culture system is set up, and time is indicated as days in vitro since co-culture (DIV - CC). Treatments are indicated the day they were performed after setting up the co-culture. B, Co-culture experimental scheme with representative phase contrast and immunocytochemistry images from WT endothelial cells (Occ: Occludin, ZO-1: Zonula occludens, H: Hoechst) and neuron-glia mixed culture (GluA1, MAP2) before setting up the co-culture. According to the time line in A , WT endothelial cells seeded in the transwell device (TW) were placed inside the neuron-glia mixed culture well. Both parts can communicate through the semipermeable membrane at the bottom of the transwell.

Article Snippet: Endothelial cells were kept in an incubator at 37°C with a humidified atmosphere with 5% CO 2 . hCMEC/D3 cells were seeded at 2 × 10 5 cells/mL and, when transwell inserts (Tw: Transwell polyethylene terephthalate transparent membrane inserts, pore size 0.4μm; Sarstedt) were used, they were additionally coated with 1% fibronectin (Sigma) in Hanks’ Balanced Salt Solution (HBSS; ThermoFisher Scientific).

Techniques: In Vitro, Co-Culture Assay, Immunocytochemistry, Membrane