Technical library

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.

Both products are Human Umbilical Vein Endothelial Cells (HUVEC) and are comparable in terms of culture conditions, passage number after thawing, and growth performance. The key differences relate to donor origin and preparation.
 
HUVEC single donor (C‑12200, C-12206)

• Isolated from one individual umbilical cord
• Expanded in primary culture and cryopreserved at subconfluency
• After thawing, cells are at passage 1 (P1)
• Represent the biological characteristics of a single donor

HUVEC pooled (C‑12203, C-12208)

• Cells are simultaneously isolated from 2-4 different umbilical cords
• Initially cultured in separate tissue‑culture dishes
• Cells are pooled after trypsinization, provided their growth rates are comparable
• After thawing, cells are also at passage 1 (P1)

Growth characteristics

• Doubling times are comparable for both products, typically 14–24 hours per population doubling
• HUVEC pooled cultures may show a slightly more heterogeneous morphology, with somewhat more elongated cells, compared to single‑donor HUVEC

Recommended culture media

Both HUVEC single donor (C-12200) and pooled HUVEC (C-12203) can be cultured in Endothelial Cell Growth Medium (C‑22010), or Endothelial Cell Growth Medium 2 (C‑22011). HUVEC 2, single donor (C‑12206), and pooled HUVEC 2 (C‑12208) are isolated directly in Endothelial Cell Growth Medium 2 and are therefore best cultured in Endothelial Cell Growth Medium 2 (C‑22011).

The heparin supplied with PromoCell Endothelial Cell Growth Media (C‑22010, C‑22011, C‑22020) is derived from 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.

To achieve high post‑thaw viability, please follow the protocol below for anchorage‑dependent primary human cells.

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

Seeding options: Proceed with one of the following options:
Option A: Direct plating

• Plate the diluted cell suspension directly at the recommended seeding density.
• Change the medium after 16–24 hours.

Option B: With centrifugation

• Centrifuge the diluted cell suspension, discard the supernatant, and resuspend the cell pellet in 1 ml of fresh medium.
• Plate the cells at the recommended seeding density.
• Change the medium no earlier than 24 hours after seeding.

Note
For cell‑type‑specific handling recommendations, please refer to the Instruction Manual of the respective cell type, as thawing and seeding requirements may vary.

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 are mixtures of proteolytic enzymes used for cell detachment and dissociation.

Trypsin

• Derived from porcine pancreas
• Commonly used for detaching anchorage‑dependent cells
• Typically requires an inactivation step (e.g. with serum or Trypsin Neutralizing Solution)

Accutase

• Derived from invertebrate sources
• Can be used to detach anchorage‑dependent cells and to reduce clumping of suspension cells, for example prior to cell counting
• Is more gentle than trypsin and generally results in higher cell viability
• Does not contain mammalian‑ or bacterially derived proteins
• Is more thermolabile than trypsin and usually does not require an inactivation step

Note
The suitability of trypsin or accutase depends on the cell type and application. Please refer to the Instruction Manual of the respective cell type for specific recommendations.

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