Primary Human Endothelial Cells and Growth Media

These products are available in the US, Canada, and select European countries.

• Primary Endothelial Cell Culture
• Cultivation Protocol for Human Endothelial Cells
• Endothelial Cell Culture System Quality Control
• Materials for Primary Endothelial Cell Culture

Primary Endothelial Cell Culture

PromoCell^® endothelial cells are isolated from large blood vessels and microvascular tissues. Shortly after isolation, Human Endothelial Cells are cryopreserved at passage 1 or passage 2 using proprietary, serum-free freezing medium, Cryo-SFM. Each cryovial contains more than 500,000 viable cells after thawing. Human Endothelial Cells from large vessels are also available as proliferating cell cultures made from cryopreserved cells that have been thawed and cultured for three days.

Cultivation Protocols for Human Endothelial Cells

• Protocol: Cultivation for Human Endothelial Cells
• Protocol: Endothelial tube formation angiogenesis assay
• Protocol: Endothelial hanging insert migration and invasion assay

Four different Endothelial Growth Media formulations have been optimized for culture of cells from distinct vascular sources, and with or without bovine hypothalamic extract (ECGS).

• Endothelial Cell Growth Medium and Endothelial Cell Growth Medium 2 (no ECGS) were developed for the in vitro cultivation of endothelial cells from large blood vessels including HUVEC, umbilical artery, aorta, coronary artery, pulmonary artery, and saphenous vein.  The media are optimized for growing primary human cells, but may also be used for bovine, porcine, murine, rabbit, elephant, caprine, canine, and rat endothelial cells.
• Endothelial Cell Growth Medium MV and Endothelial Cell Growth Medium MV2 (no ECGS) were developed for the in vitro cultivation of endothelial cells from microvascular vessels, the coronary artery, and the aorta. They are optimized for primary human cells, but may also be used for bovine, porcine, canine, murine, and rat endothelial cells.

All endothelial media are available in two formats: Medium (ready-to-use) and Medium Kit. The Medium (ready-to-use) option consists of a 500 mL bottle of Basal Medium and one vial of SupplementMix. The Medium Kit consists of a 500 mL bottle of Basal Medium and the SupplementPack (set of individual vials with pre-measured supplements) to provide the user with complete control over the media formulation. Adding the SupplementMix or the SupplementPack to the Basal Medium generates the complete medium. The Basal Medium (with or without phenol red), SupplementMix, and SupplementPacks are all also available for individual purchase.

Endothelial Growth Media formulations with recommendations for specific primary endothelial cell phenotypes

Table 1.

Endothelial Cell Growth Medium & Endothelial Cell Growth Medium 2	Endothelial Cell Growth Medium MV & Endothelial Cell Growth Medium MV2
Human Umbilical Vein Endothelial Cells (HUVEC)* Human Umbilical Artery Endothelial Cells (HUAEC) Human Pulmonary Artery Endothelial Cells (HPAEC) Human Saphenous Vein Endothelial Cells (HSaVEC)	Human Dermal Microvascular Endothelial Cells (HDMEC) Human Coronary Artery Endothelial Cells (HCAEC) Human Aortic Endothelial Cells (HAoEC) Human Pulmonary Microvascular Endothelial Cells (HPMEC) Human Dermal Lymphatic Endothelial Cells (HDLEC) Human Dermal Blood Endothelial Cells (HDBEC) Human Cardiac Microvascular Endothelial Cells (HCMEC) Human Uterine Microvascular Endothelial Cells (HUtMEC)

*Human Umbilical Vein Endothelial Cells (HUVEC)

For researchers working with Human Umbilical Vein Endothelial Cells, HUVEC isolated in standard Endothelial Cell Growth Medium or in Endothelial Cell Growth Medium 2 are available to suit specific applications. The cells are supplied either from single donors or from pooled donors (from up to four different umbilical cords).  Additionally, HUVEC are available which are pre-screened for VEGF (vascular endothelial growth factor) response.

Primary Endothelial Cell Growth Media Supplement Details

All endothelial media contain the growth factors and supplements necessary for the optimal growth of human endothelial cells (see table below). They do not contain antibiotics or antimycotics and are formulated for use in an incubator with 5% atmospheric CO₂

Final endothelial cell supplement concentrations
(after addition)

Table 2.

Component	Endothelial Cell Growth Medium	Endothelial Cell Growth Medium 2	Endothelial Cell Growth Medium MV	Endothelial Cell Growth Medium MV2
Fetal Calf Serum	0.02 mL/mL	0.02 mL/mL	0.05 mL / mL	0.05 mL / mL
Endothelial Cell Growth Supplement	0.004 mL/mL	—	0.004 mL / mL	—
Epidermal Growth Factor (recombinant human)	0.1 ng/mL	5 ng/mL	10 ng / mL	5 ng / mL
Basic Fibroblast Growth Factor (recombinant human)	1 ng/mL	10 ng/mL	—	10 ng / mL
Insulin-like Growth Factor (R3 IGF-1)	—	20 ng/mL	—	20 ng / mL
Vascular Endothelial Growth Factor 165 (recombinant human)	—	0.5 ng/mL	—	0.5 ng / mL
Ascorbic Acid	—	1 µg/mL	—	1 µg / mL
Heparin	90 μg/mL	22.5 µg/mL	90 μg / mL	—
Hydrocortisone	1 µg/mL	0.2 µg/mL	1 µg / mL	0.2 µg / mL

Preparing Supplemented Media for Primary Endothelial Cell Culture

• Thaw the endothelial cell SupplementMix or SupplementPack at 15 – 25 °C.
• Aseptically mix the supplement solutions by carefully pipetting up and down.
• Transfer the entire contents of each supplement to the endothelial cell Basal Medium.
• Close the bottle and swirl gently until a homogeneous mixture is achieved.

Note: Light flocculation may be seen upon thawing the supplements containing ECGS/heparin. This does not affect the activity. Optional: centrifuge and remove the precipitate under sterile conditions.

Storage and Stability of Endothelial Cell Products

• Upon arrival, immediately store the Basal Medium at 4 – 8 °C in the dark; do not freeze the Basal Medium. Store the SupplementMix or SupplementPack at -20 °C.
• If stored as directed, products are stable until the expiration date on the label.
• Once supplements are added to the Basal Medium, the shelf life of the complete endothelial cell medium is six weeks at 4 – 8 °C. Do not freeze the complete medium.
• For use, aliquot and prewarm only the volume of complete endothelial cell medium required for single use, and maintain the remaining medium refrigerated at 4 – 8 °C.
• After delivery, cryopreserved endothelial cells should be stored in liquid nitrogen or seeded directly.
• Proliferating endothelial cells must be processed immediately.

Note: The SupplementMix and SupplementPack are delivered thawed and can be frozen after arrival without loss of activity.

Endothelial cell culture system quality control

Human primary endothelial cells quality control

Rigid quality control tests are performed for each lot of human endothelial cells.

• Tested for cell morphology, adherence promotion, and cell viability.
• Flow cytometric analyses for cell type-specific markers are performed for each lot.
• Growth performance is tested through multiple passages up to 15 population doublings (PD) under culture conditions without antibiotics and antimycotics.
• Tested for the absence of HIV-1, HIV-2, HBV, HCV, HTLV-1, HTLV-2 and microbial contaminants (fungi, bacteria, and mycoplasma).
• A detailed certificate of analysis (CoA) for each lot is available for download.

Table 3. Endothelial Cell Specifications

Product	Recommended Culture Media	Plating Density	Passage after Thawing	Marker	Population Doublings
Human Umbilical Vein Endothelial Cells (HUVEC), single donor	C-22010 C-22011	5,000 –10,000 cells per cm²	P1	vW+ CD31+ Dil-Ac-LDL uptake+	> 15
Human Umbilical Vein Endothelial Cells (HUVEC), pooled	C-22010 C-22011	5,000 –10,000 cells per cm²	P1	vWF+ CD31+ Dil-Ac-LDL uptake+	> 15
Human Umbilical Vein Endothelial Cells (HUVEC) isolated in Growth Medium 2, single donor	C-22011	5,000 –10,000 cells per cm²	P1	vWF+ CD31+ Dil-Ac-LDL uptake+	> 15
Human Umbilical Vein Endothelial Cells (HUVEC) isolated in Growth Medium 2, pooled	C-22011	5,000 –10,000 cells per cm²	P1	vWF+ CD31+ Dil-Ac-LDL uptake+	> 15
Human Umbilical Vein Endothelial Cells (HUVEC), pre-screened	C-22010 C-22011	5,000 –10,000 cells per cm²	P1	vWF+ CD31+ Dil-Ac-LDL uptake+ VEGF response	> 15
Human Umbilical Artery Endothelial Cells (HUAEC)	C-22010 C-22011	5,000 –10,000 cells per cm²	P1	vWF+ CD31+ Dil-Ac-LDL uptake+	> 15
Human Aortic Endothelial Cells (HAoEC)	C-22020 C-22022	5,000 –10,000 cells per cm²	P2	vWF+ CD31+ Dil-Ac-LDL uptake+	> 15
Human Coronary Artery Endothelial Cells (HCAEC)	C-22020 C-22022	5,000 –10,000 cells per cm²	P3	vWF+ CD31+ Dil-Ac-LDL uptake+	> 15
Human Pulmonary Artery Endothelial Cells (HPAEC)	C-22010 C-22011	5,000 –10,000 cells per cm²	P2	vWF+ CD31+ Dil-Ac-LDL uptake+	> 15
Human Saphenous Vein Endothelial Cells (HSaVEC)	C-22010 C-22011	5,000 –10,000 cells per cm²	P2	vWF+ CD31+ Dil-Ac-LDL uptake+	> 15
Human Dermal Microvascular Endothelial Cells (HDMEC), juvenile foreskin	C-22020 C-22022	10,000 - 20,000 cells per cm²	P2	vWF+ CD31+ Dil-Ac-LDL uptake+	> 15
Human Dermal Microvascular Endothelial Cells (HDMEC), adult donor	C-22020 C-22022	10,000 - 20,000 cells per cm²	P2	vWF+ CD31+ Dil-Ac-LDL uptake+	> 15
Human Dermal Microvascular Endothelial Cells (HDMEC), pre-screened	C-22020 C-22022	10,000 - 20,000 cells per cm²	P2	vWF+ CD31+ Dil-Ac-LDL uptake+ VEGF response+	> 15
Human Dermal Blood Endothelial Cells (HDBEC), juvenile foreskin	C-22020	10,000 - 20,000 cells per cm²	P2	Podoplanin- CD31+	> 15
Human Dermal Blood Endothelial Cells (HDBEC), adult donor	C-22020	10,000 - 20,000 cells per cm²	P2	Podoplanin- CD31+	> 15
Human Dermal Lymphatic Endothelial Cells (HDLEC), juvenile foreskin	C-22022	10,000 - 20,000 cells per cm²	P2	Podoplanin+ CD31+	> 15
Human Dermal Lymphatic Endothelial Cells (HDLEC), adult donor	C-22022	10,000 - 20,000 cells per cm²	P2	Podoplanin+ CD31+	> 15
Human Cardiac Microvascular Endothelial Cells (HCMEC)	C-22020 C-22022	10,000 - 20,000 cells per cm²	P2	vWF+ CD31+ Dil-Ac-LDL uptake+	> 10
Human Pulmonary Microvascular Endothelial Cells (HPMEC)	C-22020 C-22022	10,000 - 20,000 cells per cm²	P2	vWF+ CD31+ Dil-Ac-LDL uptake+	> 15
Human Uterine Microvascular Endothelial Cells (HUtMEC)	C-22020 C-22022	10,000 - 20,000 cells per cm²	P2	vWF+ CD31+ Dil-Ac-LDL uptake+	> 15

Endothelial Cell Growth Medium Quality Control

All lots of Endothelial Cell Growth Media are subjected to comprehensive quality control tests using primary human endothelial cells. Each lot is checked for growth promoting activity, adherence rate, and typical morphology of the tested endothelial cells. Approved in-house lots of endothelial cell growth media are used as a reference. In addition, all lots of media have been tested for the absence of microbial contaminants (fungi, bacteria, mycoplasma).

Intended Use for Endothelial Cells and Growth Medium

These products are for in vitro use only and not for diagnostic or therapeutic procedures. For safety precautions, please see appropriate MSDS.

Note: Due to their low serum content or the absence of serum, PromoCell media are not suitable for trypsin neutralization (when subculturing the cells, for example).  Instead we recommend using our DetachKit (C-41200, C-41210, C-41220), which contains HEPES BSS, Trypsin/EDTA and Trypsin Neutralizing Solution.

Materials for Human Primary Endothelial Cell Culture