Technical library

PromoCell's HDLEC are isolated from skin (dermis). When isolated from juvenile donors (C-12216), the exact localization is foreskin. When derived from adult donors (C-12217), the localization depends on the type of surgery, e.g. breast or temple. You can find the information on the exact localization in the Certificate of Analysis.

Our HDLEC are tested to be positive for CD31, podoplanin, and ac-LDL uptake and are delivered in P2. The recommended culture medium is Endothelial Cell Growth Medium MV2 (C-22022).

PromoCell's Endothelial Cell Growth Medium (C-22010) is a complete medium that can be used for the culture of HUVEC after addition of the SupplementMix. Addition of extra FCS is not necessary. The SupplementMix contains FCS (2% v/v final concentration), recombinant growth factors, hormones, and a bovine brain extract that together have mitogenic effects on endothelial cells.

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).

The source of our heparin is ex porcine mucosa.

Generally, the medium should be changed every 2-3 days. Please note: Following thawing, the first medium change should be performed after 16-24 hours to prevent cell damage due to residual freezing medium.

• Remove the cryovial from liquid nitrogen and transport it on dry ice to the cell culture laboratory.
• Thaw the cells for 2 minutes at 37 °C until the contents are just defrosted. During thawing, keep the vial immersed in the water bath up to, but not above, the screw cap.
• Under a laminar flow hood, carefully disinfect the vial thoroughly with 70% ethanol.
• Transfer the thawed cell suspension into 9 ml of pre‑warmed culture medium (1:10 dilution).
• Proceed with one of the following options:

For mineralization assays, HOB are cultured in Osteoblast Mineralization Medium (C-27020). Mineralization can be detected after approximately 3 weeks by incorporation of Ca-45, or it can be visualized by von Kossa or Alizarin Red staining for calcium.

Yes, it is possible. Short protocol: Plate human osteoblasts in Osteoblast Mineralization Medium on collagen I coated TC vessels. Incubate the cells for 17-21 days and change the medium every third day. Be careful not to disturb the cell monolayer. Fix the cells. The calcium deposition can be visualized by von Kossa or Alizarin Red staining. More detailed information on osteoblast mineralization and Alizarin Red S staining can be found in the attached Application Note.

Our HCASMC (C-12221) are isolated from the large arteries, i.e. from

• Right coronary artery
• Left main coronary artery
• Circumflex coronary artery and
• Left anterior descending coronary artery.

Both, trypsin and accutase represent mixtures of different proteolytic enzymes. Trypsin is prepared from porcine pancreas, accutase from invertebrates. Accutase can replace trypsin for the detachment and dissociation of anchorage-dependent cells from surfaces and can also be used on suspension cells to reduce clumping in preparation for counting. The advantages of accutase over the traditional trypsin treatment are that it is more gentle and less damaging to cells (leading to increased viability) and does not contain any mammalian or bacterially derived proteins.

Accutase is more thermolabile than trypsin and usually doesn't require an inactivation step.