Umbilical cord

WJ-MSC/TERT273

Mesenchymal stem cells play an essential role in tissue homeostasis and repair, whereby evidence has accumulated that these effects are at least in part mediated by secreted extracellular vesicles (EVs). To boost the development of EV-based therapeutics, continuously growing, standardizable EV production hosts are of ever-increasing importance. Evercyte ́s WJ-MSC/TERT273 cell line fulfils all requirements for production of clinical grade EVs under GMP conditions. EVs secreted from our WJ-MSC/TERT273 cell line show neo-angiogenic, anti-inflammatory, anti-fibrotic and wound healing activities as demonstrated by in vitro bioassays. Additionally, the cells are valuable for studying processes such as differentiation, inflammation, tissue homeostasis and repair.

General information

Cat#: CHT-021-0273

Organism: homo sapiens
Tissue, cell type: Wharton´s Jelly / umbilical cord, mesenchymal stem cells
Morphology: mesenchymal, spindle-shaped 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 growth characteristics

WJ-MSC/TERT273 cells can be grown for a minimum of 50 population doublings with a stable growth rate and without showing signs of growth retardation. The cells are characterized by the typical spindle-shaped mesenchymal morphology.

Expression of MSC marker proteins CD73, CD90, CD105

WJ-MSC/TERT273 cells homogeneously express typical marker proteins of mesenchymal stem cells such as CD73, CD90 and CD105 (green peaks). The cells do not express CD34 (green peaks) as shown by indirect immunofluorescence stainings. Cells stained with the corresponding isotype-control antibodies were used as negative controls (red peaks).

Characterization of extracellular vesicles / cryoEM and western blot

Extracellular vesicles from WJ-MSC/TERT273 cells show the characteristic lipid double layer membrane (cryo-EM) and typical marker proteins such as syntenin and CD81 are detected, whereas calnexin is only present in cell lysates (western blotting).

Production of extracellular vesicles and miRNA cargo of EVs

Extracellular vesicles from WJ-MSC/TERT273 are produced in a hollow fiber bioreactor system with continuous harvests over a peroid of at least 4 months. Characterization of EVs harvested at different time points reveals stable miRNA cargo throughout the whole production process.

Influence of WJ-MSC/TERT273 derived EVs on myofibroblast differentiation

Treatment of telomerized human dermal fibroblasts fHDF/TERT166 with transforming growth factor beta (TGF-ß) induces expression of alpha smooth muscle actin (𝛂-SMA).
Treatment of cells with TGF-ß together with extracellular vesicles (EVs) from Wharton´s Jelly derived mesenchymal stem cells (WJ-MSC/TERT273) reduces 𝛂-SMA expression significantly, demonstrating an effect of EVs on myofibroblast differentiation.

Fibroblast growth promoting activity of WJ-MSC/TERT273 derived EVs

fHDF/TERT166 were seeded into chamber slides and a physical gab within the monolayer was created followed by monitored the process of cell migration into the gap. Significantly less free area between the cells was detected upon addition of extracellular vesicles from MSCs (WJ-MSC/TERT273).

Neo-angiogenic potential of WJ-MSC/TERT273 derived EVs

Treatment of endothelial cell spheroids embedded in a semi-solid matrix with extracellular vesicles from WJ-MSC/TERT273 cells induces sprout formation similar as vascular endothelial growth factor (VEGF) indicating neo-angiogenic properties of EVs.

Anti-inflammatory activity of WJ-MSC/TERT273 derived EVs

Treatment of mouse macrophage cells with lipopolysaccharide (LPS) induces an inflammatory reaction as mirrored by formation of nitric oxide (NO).
Addition of extracellular vesicles from WJ-MSC/TERT273 cells significantly reduces NO formation indicating an anti-inflammatory activity of the EVs.

FAQs

In vitro propagation

Mesencult – ACF Plus Culture Kit

Mesencult – ACF Plus Culture Kit including Animal Component-Free Cell Attachment  (STEMCELL TECHNOLOGIES, Cat# 05448)

2 mM GlutaMAXTM-I (Gibco, Cat# 35050-038)

200 µg/ml G418 (InvivoGen, Cat# ant-gn5)

Additional material & reagents

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

CTS TrypLE Select Enzym (Gibco, Cat# A1285901)

Passaging of cells

The new culture flasks have to be pre-coated with ACF Cell Attachment Substrate following the instructions of the manufacturer. Briefly, dilute the substrate 1:300 in PBS and transfer the diluted substrate to the culture flasks (72 µl/cm²), incubate at least 2 hours at room temperature.

Before introducing cells, remove excess of ACF substrate and rinse flask once with PBS (160 µl/cm²).
Then, add CTS TrypLE Select Enzym 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 2-3 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 growth medium (about 160 µl/cm²) and centrifuge at 300 g for 5 min
Discard the supernatant, resuspend the cell pellet in the remaining droplet and add growth medium (about 160 µl/cm²).

Then, transfer appropriate aliquots of the cell suspension to new culture vessels supplemented with growth medium (final volume of 240 µl/cm²).
A split ratio of 1:3 – 1:4 twice a week is recommended (after cells have reached about 80-90 % confluence).

Cultivate cells at 37°C in a humidified atmosphere with 5% CO2.

Cryopreservation

Freezing medium

CryoStor(R) cell cryopreservation medium CS10 (Sigma Aldrich, Cat# C2874

Additional material & reagents

Phosphate buffered saline (PBS) (Sigma, Cat# D8537)
CTS TrypLE Select Enzym (Gibco, Cat# A1285901)

Freezing of cells

Detach the cells after having reached about 80-90 % confluence from the culture vessel by using CTS TrypLE Select Enzym solution (Protocol passaging of WJ-MSC/TERT273).

Resuspend the detached cells in growth medium and centrifuge at 300 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 roux flask

Pre-coat a cm² roux flask with ACF Cell Attachment substrate (Protocol passaging of WJ-MSC/TERT273).

Add 6 ml of complete growth medium to a pre-coated 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 about 80 % confluent at this point, they have to be passaged.

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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WJ-MSC/TERT273
Cat#: CHT-021-0273

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FOR PROFIT INDUSTRY

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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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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.

Print Friendly, PDF & Email

from

€ 1300,–

WJ-MSC/TERT273
Cat#: CHT-021-0273

ONLY FOR NON PROFIT

FOR PROFIT INDUSTRY

FOR PROFIT-CRO