Technical library

Microbial contamination is typically introduced during cell culture handling and may originate from several common sources.
 
Typical causes include:

• Unsterile working techniques (e.g. insufficient disinfection, improper aseptic handling)
• Contaminated equipment or environment such as:
  • Water in water baths
  • Incubators
  • Laminar flow hoods
• Contaminated reagents or consumables including:
  • Culture media or supplements
  • PBS, trypsin, or other solutions
  • Cell culture plasticware

Microbial contamination can severely affect cell growth and morphology.

Your observation can be either ascribed to a change of the culture medium, to a cross-contamination with another cell type, or to differentiation or senescence. The attached trouble shooting guide should help you to identify the reason for your observation.

Normal cells have a finite life span and therefore eventually stop growing and become senescent. In addition, a cease in proliferation or cell death can be induced by other factors. For more details, check the trouble shooting guide below.

In principle, all microvascular endothelial cells should be able to migrate, proliferate and form tubes or sprouts in an appropriate assay after angiogenic stimulation. As this is not part of our routine quality control procedure, we cannot tell for sure whether all cell lots will respond to angiogenic stimuli. However PromoCell also supplies HDMEC pre-screened (C-12215) that are especially tested for a positive VEGF response.

No, we don't determine the ratio of HDLEC and HDBEC in our HDMEC lots. From our experience, the percentage of HDLEC is highly lot-dependent and can vary between 5 and 60%.

Our HDMEC are isolated from the dermis of juvenile foreskin or adult skin. The purity is > 95%. Since the dermis contains blood and lymphatic capillaries, HDMEC cultures comprise blood and lymphatic microvascular endothelial cells that have differing morphologies. Both cell types have a common origin and can be identified by several markers. The ratio of lymphatic and blood derived endothelial cells can vary from lot to lot and is not determined at PromoCell.

• FBS (or BSA) can be substituted for HSA, however we do not recommend this as the FBS will lead to unpreferable immunologic stimulation of human macrophages.

• The addition of EDTA to the PBS is to further augment the “anti-clumping” activity of the Ca²⁺/Mg²⁺ free PBS.

A concentration > 10% FBS is needed to completely inactivate the trypsin. As most of PromoCell's growth media are serum-reduced or serum-free, the use of a trypsin inhibitor like TNS is highly recommended.

The standard medium recommended by PromoCell for the isolation and expansion of HUVEC, HUAEC, HPAEC, and HSaVEC is Endothelial Cell Growth Medium (C‑22010).
 
Endothelial Cell Growth Medium (C‑22010)

• Contains ECGS (Endothelial Cell Growth Supplement), an extract derived from bovine hypothalamus
• ECGS has mitogenic activity and robustly supports endothelial cell proliferation
• Widely used as a classical, well-established endothelial cell culture medium
• Contains 2% FBS

Endothelial Cell Growth Medium 2 (C‑22011)
For researchers who prefer a more defined formulation, PromoCell offers Endothelial Cell Growth Medium 2. 

• In Endothelial Cell Growth Medium 2, ECGS is replaced by defined recombinant growth factors, including VEGF and IGF, as well as higher levels of bFGF and EGF than in C-22010.
• Like Endothelial Cell Growth Medium, Endothelial Cell Growth Medium 2 also contains 2% FBS.
• To support users who routinely culture endothelial cells in Endothelial Cell Growth Medium 2, PromoCell also offers Growth Medium 2-adapted cells: HUVEC 2, single donor (C‑12206) and pooled HUVEC 2 (C‑12208) are isolated directly in Endothelial Cell Growth Medium 2.

Summary

• Standard choice: Endothelial Cell Growth Medium (C‑22010)
• More defined alternative: Endothelial Cell Growth Medium 2 (C‑22011)
• FBS content: Both media are low-serum media containing 2% FBS
• Both media are suitable for HUVEC culture. The choice depends primarily on whether a classical ECGS-based formulation or a more defined recombinant growth factor composition is preferred.

PromoCell Human Dermal Lymphatic Endothelial Cells (HDLEC) are isolated from the dermis of human skin and are delivered at passage 2 (P2).

Tissue localization

• Juvenile donors (C‑12216): Cells are isolated from foreskin.
• Adult donors (C‑12217): The exact localization depends on the type of surgery (e.g. breast, temple, labia).

Detailed information on the specific tissue origin of each cell lot is provided in the Certificate of Analysis (CoA).

Cell characterization
PromoCell HDLEC are quality‑controlled and tested positive for:

• CD31
• Podoplanin
• Ac‑LDL uptake

Recommended culture medium
The recommended medium for culturing HDLEC is Endothelial Cell Growth Medium MV2 (C‑22022).