Vicell analyzer

We count a total cell count in each passage using a Z2-Counter (Beckman Coulter, Miami, FL, USA). For each measurement, we analyzed 100 µL/1 mL cell suspension (approximately 1.5 × 10⁶ cells/1 mL). Proliferation capacity was evaluated as population doublings (PDs) and population doubling time in hours (Equation (1)). Proliferation activity was compared from the 1st passage when hNDP-SCs were seeded into two different growth media.

Equation (1). N_x is the total passage cell count calculated using the Z2-Counter (Beckman Coulter), and N₁ is the initial cell count seeded into the culture dish (5000 cells/cm²).
To determine the number of viable hNDP-SCs cells from each sample in the 3rd and 11th passage, we used the trypan dye exclusion method by Vi-Cell analyzer (Beckman Coulter). This method is based on the principle that viable cells do not take up the trypan blue dye, whereas non-viable cells do due to disturbed cell membrane. For each analysis, we utilized 250 µL of cell suspension (approximately 1.5 × 10⁶ cells/1 mL) and 250 µL PBS.

Vehicle- and MEDI3039-treated (10 pM, 24 h) cells (n = 3) were harvested, counted and viability assessed using a Vi-CELL analyzer (Beckman). The cells were then resuspended in HBS buffer and incubated with [¹⁸F]FPenM-C2Am (1–10 μM, 7–11 MBq, Am = ~ 10.5 GBq/µmol at the start of labeling) at 37 °C for 20 min. Cell pellets (5 million cells, centrifuged at 700g for 5 min at 4 °C) were washed three times with HBS buffer (1 mL) and radioactivity counted for 1 min using a gamma counter (AMG Hidex) set to monitor the fluorine-18 gamma emission (511 keV).

Primary Human Umbilical Vein Endothelial Cells (HUVEC) were isolated from umbilical cords of healthy neonates directly after cesarean delivery at the Department of Gynecology and Obstetrics, University Hospital, LMU Munich, Germany. Written informed consent from the mother was obtained before donating umbilical cords in accordance with the Declaration of Helsinki. After collagenase A (Roche) treatment, HUVEC were detached from umbilical vein vascular wall and cultured in Endothelial Cell Basal Medium (ECGM; PromoCell) with Supplement Mix (PromoCell), supplemented with 10% FCS (Biochrom AG) and 1% Penicillin/Streptomycin (Pen/Strep, Gibco). A ViCell analyzer (Beckman Coulter, Fullerton, CA, United States) was used to evaluate the cell count and viability. For each of the independent experiments, we used non-pooled cells obtained from different donors at passage 2–4.