Technical library - RESOURCES

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.

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. 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.

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.

We recommend to use the trypsin as well as the other detach solutions at room temperature to avoid overtrypsinization and irreversible cellular damage.

Upon arrival, the basal media should be stored between 4°C and 8°C, the supplements at -20°C.

Please do not freeze our cell culture media. Freezing can lead to irreversible precipitation of media components and the quality can no longer be assured.

Yes, you can use accutase to detach Normal Human Cells. Accutase acts very gently on the cells. Cell membranes and surface epitopes will not be harmed. It is therefore mostly used for applications that require unchanged surface markers, e.g. for flow cytometry, or for detachment of very sensitive cells.