Our HUVEC single donor (C-12200) are isolated from a single umbilical cord; propagated in primary culture; and frozen down at subconfluency.
For the preparation of HUVEC-pooled (C12203); we simultaneously isolate the cells from 2-4 umbilical cords and grow them in separate tissue culture dishes. The cells are pooled after trypsinization given that their growth rates are comparable.
After thawing; our HUVECs (single donor and pooled) are both in P1. The recommended media are Endothelial Cell Growth Medium (C-22010) or Endothelial Cell Growth Medium 2 (C-22011). With respect to cell growth; HUVEC-pooled tend to have a more heterogeneous morphology with slightly more elongated cells but the doubling times are comparabel for both types (typically 18-36 hrs per doubling).
A subconfluent T25-flask typically contains between 0.9 and 1.2 million cells corresponding to 36;000-48;000 cells per cm2. It is recommended to count the existing cell number after trypsinization and to calculate the needed number of new flasks.
Recommended seeding density for HUVEC is 5;000-10;000 cells/cm2. This usually corresponds to a split ratio of 1:4-1:6. 1:6 means that you can increase the culture surface by factor 6 (e.g. from 1x T25 to 6x T25 or 2x T75).