Human Umbilical Vein Endothelial Cells 2 (HUVEC 2)

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Some of our customers have successfully used PromoCell Endothelial Cell Growth Medium MV2 (C-22022) when co-culturing Human Coronary Artery Endothelial Cells (HCAEC; C-12221) and Normal Human Dermal Fibroblasts (NHDF; C-12300).

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).

Short protocol:

• Trypsinize the cells as usual
• Centrifuge and resuspend in suitable cold freezing medium at a density of 1-4 x 10⁶ cells/ml
• Cool down the cells slowly to -80°C (approx. -1°C per min). We recommend to use "Mr. Frosty" from Nalge or "CoolCell" from Biocision; which both provide gradual and controlled cooling rates when placed in a -80°C freezer overnight.
• Transfer the vials into liquid nitrogen for long-term storage

The standard medium for isolation and propagation of our HUVEC HUAEC HPAEC and HSaVEC is Endothelial Cell Growth Medium (C-22010). It contains ECGS an extract from bovine hypothalamus which has mitogenic effects on endothelial cell proliferation. Scientists who prefer a more defined Growth Medium can use Endothelial Cell Growth Medium 2 (C-22011). In this medium ECGS is replaced by VEGF IGF and additional bFGF and EGF to stimulate endothelial cell growth.

• Mesenchymal Stem Cells (C-12974/C-12971/C-12977) need Fibronectin-coating when grown in PromoCell MSC Growth Medium XF (C-28019) and when differentiated in MSC Neurogenic (C-28015); Adipogenic (C-28016); or Osteogenic (C-28013) Differentiation Media.
• Human monocyte-derived macrophages (C-12914/C-12916/C-12915/C-12917) must be seeded into Fibronectin-coated culture vessels in combination with PromoCell's M1- and M2-Generation Media XF (C-28055; C-28056).
• For efficient induction of osteoblast mineralization with PromoCell's Osteoblast Mineralization Medium (C-27020); the TC plates should be pre-coated with collagen type I.

• Liquid phase storage provides a consistent temperature of -196°C; a longer holding time and a greater vial capacity but involves the risk of contamination issues.
• Storage in the gas phase is very safe with respect to contaminations but the holding time of the cells is shorter and the vial capacity is reduced.

General protocol for recovery of anchorage-dependent primary cells: Remove vial from liquid nitrogen; transport it on dry ice to the cell culture lab. Thaw in a 37°C waterbath for approx. 2 min; until it is just defrosted. Keep the vial immersed in water until just below the screw cap during thawing and only remove it shortly after approx. 90 sec to check the progress. Do not repeatedly insert and remove the vial while thawing in water. Carefully disinfect the vial with plenty of 70% EtOH under the laminar flow hood and aseptically transfer the thawed suspension into an appropriate TC dish with growth medium (pre-warmed in the incubator for > 30 min). The cells usually attach within a few hours. Perform media change after 24 hrs at the latest to remove residual DMSO from the freezing media. Additional information can be found in the instruction manuals of our cells.

The study of angiogenesis has been significantly advanced by the ability to culture endothelial cells in vitro. Initially large vessel ECs such as those isolated from the human umbilical vein (HUVEC) were used for these studies but increasingly it has been recognized that microvascular endothelial cells are a more appropriate model since angiogenesis involves microvessels rather than large vessel ECs.

The amount of media needed per vial depends on the growth characteristics of the cells the size of the TC vessels and the split ratios used the frequency of media changes the type of experiments you perform etc. It is therefore difficult to give definite quantities. As a rough guideline 1-2 bottles (500 ml each) are needed for 1 vial of HUVEC.