Skin – vascular system

HDMVEC/TERT164-B

Evercyte ́s human dermal microvascular endothelial cell line HDMVEC/TERT164-B can be grown without limitations while maintaining expression of cell type specific markers and function. Therefore, these cells are useful for setting up in vitro bioassays for studying angiogenesis in disorders involving the lymphatic system such as wound healing or metastasis as well as bioassays for testing the angiogenic properties of novel drugs. Additionally, the cell line is the perfect starting material for genetic engineering to create important disease models.

General information

Cat#: CHT-013-0164-B

Organism: homo sapiens
Tissue, cell type: Skin (female, 52 years), microvascular endothelial cells, lymphatic
Morphology:
endothelial morphology
Life span extension:
ectopic expression of hTERT
Quality:
free from contaminations (bacteria incl. mycoplasma, fungi, HIV, HAV, HBV, HCV, Parvo-B19) and cross-contaminations

Morphology and differentiation on Matrigel

HDMVEC/TERT164-B cells can be grown for a minimum of 50 PDs with a stable growth rate. The cells are characterized by the typical endothelial morphology and form tubule-like structures when inoculated onto matrigel matrix indicating neo-angiogenic properties.

Expression of marker proteins

HDMVEC/TERT164-B cells homogenously express typical marker proteins of lymphatic microvascular endothelial cells such as von Willebrand factor (vWF), CD105, podoplanin and CD31 as demonstrated by immunofluorescence stainings (cell nuclei are counterstained with DAPI / left pictures; cells stained with isotype-control antibody as negative control / right picture).

PAN induced toxicity – 3D co-culture with podocytes

HDMVEC/TERT164 cells, co-cultured with telomerised podocytes PODO/TERT256 in transwells, form a barrier that retains bovine serum albumin (BSA); puromycin aminonucleoside (PAN) disrupts this barrier; mizoribin (MRZ) can rescue PAN induced injury.

FAQs

In vitro propagation

Endopan MV Kit (PAN Biotech) supplemented with G418

Endopan MV basalmedium (PAN Biotech, Cat# P04-0020B)

Endopan MV supplements (PAN Biotech, Cat# P04-0020S)

20 µg/ml G418 (InvivoGen, Cat# ant-gn-5)

Additional material & reagents

0,1 % Gelatin (Sigma, Cat# G1393, 2 %), dilute in PBS

Phosphate buffered saline (PBS) (Sigma, Cat# D8537)

0,05 % Trypsin-EDTA (Gibco, Cat#25300-054)

Defined Trypsin-Inhibitor (Gibco, Cat# R007100)

Protocol passaging of HDMVEC/TERT164-B
Passaging of cells

The new culture flasks have to be pre-coated with gelatin. Therefore, the culture flasks are treated with 0.1 % gelatin solution (80 µl/cm²) at 37°C for at least 10 min (10 – 60 min). Before introducing cells, remove excess of gelatin solution. Use the culture flask immediately for seeding the cells, the surface must not dry out.

For detachment of the cells remove and discard the culture medium and wash the cells twice with PBS (each 160 µl/cm²). Remove PBS completely.

Then, add 0.05 % Trypsin-EDTA solution (20 µl/cm²), make sure that all cells have been in contact with this solution and incubate the culture flask at 37°C for approximately 3 min.
Resuspend the cells in growth medium (about 160 µl/cm2) and centrifuge at 170 g for 5 min.
Observe cell detachment under an inverted microscope. As soon as all cells are detached (if necessary, agitate the cells by gently hitting the flask), add Trypsin-Inhibitor (20 µl/cm²).
Thereafter, resuspend the cells in growth medium (about 160 µl/cm²) and aspirate the cells by pipetting, centrifuge at 170 g for 5 min.
Discard the supernatant, resuspend the cell pellet in the remaining droplet and add growth medium. Then, add appropriate aliquots of the cell suspension to gelatin coated culture vessels supplemented with growth medium (final volume of 240 µl/cm²).
A split ratio of 1:2 to 1:3 twice a week is recommended (after cells have reached about 90 % confluence). Perform a medium change after 2-3 days if cells have not reached required cell density.
Cultivate cells at 37°C in a humidified atmosphere with 5% CO2.
Aliquot complete Endopan MV medium and store at 4°C for a maximum of 4 weeks at 4°C, temper the medium to room temperature (not 37°C) before use.

Cryopreservation

Freezing medium

Endopan MV complete medium (PAN Biotech, Cat# P04-0020B+P04-0020S)

10 % Fetal bovine serum (Sigma Aldrich, Cat# F7524)

10 % DMSO (Sigma Aldrich, Cat# D2650)

Additional material & reagents

Phosphate buffered saline (PBS) (Sigma, Cat# D8537)

0,1 % Gelatin (Sigma, Cat# G1393, 2 %), dilute in PBS

0,05 % Trypsin-EDTA (Gibco, Cat#25300-054)

Defined Trypsin-Inhibitor (Gibco, Cat# R007100)

Protocol cryopreservation of HDMVEC/TERT164-B
Freezing of cells

Detach the cells from the culture vessel by using Trypsin-EDTA solution (Protocol passaging of HDMVEC/TERT164-B).

Resuspend the detached cells in growth medium Endopan MV complete medium and centrifuge at 170 g for 5 min.
Discard the supernatant, resuspend the resulting cell pellet in the remaining droplet and add freezing medium (tempered to 4°C) to reach a cell density of about 5 x 105 cells/ml (for thawing in a 25 cm² culture flask).
Add 1 ml of this cell suspension to each pre-cooled cryovial and immediately transfer the cells to -80°C.
After 24 hours transfer the vials to the liquid nitrogen tank.
Thawing of cells

Original Evercyte cells are to be thawed in a T25 rouxflask

Before thawing the original vial containing Evercyte cells, pre-coat a 25 cm² culture flask with gelatin (Protocol passaging of HDMVEC/TERT164-B).
Add 6 ml of complete growth medium Endopan MV to a 25 cm² culture flask and place the culture flask in the incubator for at least 30 min to allow the medium to reach its normal pH.
Take a vial of frozen cells, rinse it outside with Ethanol and pre-warm in the hand until one last piece of frozen cells is seen.
Then, immediately transfer the content of the vial to a 15 ml centrifugation tube pre-filled with 9 ml of medium pre-cooled to 4°C and centrifuge for 5 min at 170 g.
Discard the supernatant and resuspend the cell pellet in the remaining droplet.
Add 1 ml of the pre-warmed medium to the cells, transfer them to the prepared culture flask and incubate at 37°C in a suitable incubator.
Perform a medium change 24 hours after thawing. If the cells are already 80 % confluent at this point, they have to be passaged.
Protocol passaging of HDMVEC/TERT164-B

Product data sheet – certificate of analysis

Product data sheet (PDS) download
Certificate of analysis
is available upon request | Please contact us indicating the respective LOT numbers

 

Protocols

Preparation of cell culture medium Endopan MV
Protocol passaging of HDMVEC/TERT164-B
Protocol cryopreservation of HDMVEC/TERT164-B
Protocol for differentiation of HDMVEC/TERT164-B on Matrigel
Protocol for immunofluorescent staining of HDMVEC/TERT164-B for marker expression

Data on Markers and Functions

HDMVEC/TERT164-B – morphology and differentiation on Matrigel
HDMVEC/TERT164-B – expression of vWF, CD105, Podoplanin, CD31
HDMVEC/TERT164-B – glomerular barrier model / 3D coculture with podocytes (PODO/TERT256)

 

Safety documents (coming soon)

Telomerized human cells – material safety data sheet
HDMVEC/TERT164-B – cell line establishment, vector maps

Selected publications

Gludovacz E, et al. (2020) Human diamine oxidase cellular binding and internalization in vitro and rapid clearance in vivo are not mediated by N-glycans but by heparan sulfate proteoglycan interactions.  Glycobiology, cwaa090. https://pubmed.ncbi.nlm.nih.gov/32985651.

Licence Conditions

The business concept of Evercyte is to out-license telomerized cells to our customers. The license conditions depend on whether the contract partner is a for profit or a nonprofit organization and the intended use of the cells.

Nonprofit organizations

Evercyte grants licenses for an unlimited period to academic or nonprofit-organizations, whereby the use of Evercyte cell lines is restricted to research & development purposes and non-commercial use. The cells are not intended for human use.
The customers have to agree to the conditions described in our material transfer agreement as well as accept our general terms and conditions.
On time payment for unlimited use: EUR 1300

Profit organizations

Pharmaceutical – chemical- cosmetic industries
Evercyte grants licenses for commercial organizations, whereby we offer an initial testing phase for a flat fee that allows our customers to test our cells in their laboratories for a period of 6 months.
Thereafter, annual license fees fall due, depending on the cell line of interest. Besides offering cell lines for research & development purposes, we also have established cell factories that qualify for production of clinical grade extracellular vesicles for human application.
The customer has to agree to the conditions described in our license agreements.
Contract research organizations (CRO)
Evercyte grants licenses for contract research organizations, whereby we offer an initial testing phase for a flat fee that allows our customers to test our cells in their laboratories. Thereafter, we would negotiate a royalty based long-term license agreement individually.
The use of the cells during these phases is restricted to research & development purposes. The cells are not intended for human use. The customers have to agree to the conditions described in our material transfer agreement and accept our general terms and conditions.
Initial license fee for 6 months: EUR 2000
Annual license fee R&D: royalty based
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“I have had the pleasure of working with Evercyte for the last few years. We continually rely on Evercyte because of the high-quality data that they produce, their diligent responsiveness, and their excellent customer service.”

 

Josh Garlich, Senior Research Scientist, Apellis Pharmaceuticals, Inc.

“Cytonus has been working with Evercyte from many years as they are a trusted partner and have always delivered the highest quality cell lines to advance our platform.  We routinely draw on their expertise to meet cellular engineering challenges and they have not disappointed.”

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Customer Reviews

“I have had the pleasure of working with Evercyte for the last few years. We continually rely on Evercyte because of the high-quality data that they produce, their diligent responsiveness, and their excellent customer service.”

 

Josh Garlich, Senior Research Scientist, Apellis Pharmaceuticals, Inc.

“Cytonus has been working with Evercyte from many years as they are a trusted partner and have always delivered the highest quality cell lines to advance our platform.  We routinely draw on their expertise to meet cellular engineering challenges and they have not disappointed.”

Remo Moomiaie-Qajar, Cytonus Therapeutics, Inc.

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from

€ 1300,–

HDMVEC/TERT164-B
Cat#: CHT-013-0164-B

Ordering

ONLY FOR NON PROFIT

Ordering

FOR PROFIT INDUSTRY

Ordering

FOR PROFIT-CRO