ASC52telo, hTERT immortalized adipose derived Mesenchymal stem cells (ATCC® SCRC-4000)

Organism: Homo sapiens, human  /  Cell Type: Mesenchymal stem cells immortalized with hTERT  /  Tissue: adipose tissue  / 

Permits and Restrictions

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Organism Homo sapiens, human
Tissue adipose tissue
Cell Type Mesenchymal stem cells immortalized with hTERT
Product Format frozen
Morphology fibroblast-like
Culture Properties adherent
Biosafety Level 2  [Cells contain SV40 viral DNA sequences]
Gender female
Ethnicity Caucasian
Storage Conditions liquid nitrogen vapor phase
Clinical Data
female
Caucasian
Antigen Expression
Positive surface markers: CD29, CD44, CD73, CD90, CD105, CD166 (>90%)

Negative surface markers: CD14, CD19, CD34, CD45 (<5%)

Genes Expressed
CD29+ (verified at ATCC), CD44+ (verified at ATCC), CD73+ (verified at ATCC),CD90+ (verified at ATCC),CD105+ (verified at ATCC), CD166+ (verified at ATCC)
Complete Growth Medium

The base medium for this cell line is Mesenchymal Stem Cell Basal Medium (PCS-500-030). To make the complete growth medium, add Mesenchymal Stem Cell Growth Kit (PCS-500-040) for Adipose and Umbilical-derived MSCs - Low Serum  Components and G418  to the base medium as the following:

485 mL of basal medium (PCS-500-030)

10 mL of MSC supplement (2% FBS, 5 ng/mL rh FGF basic, 5 ng/mL rh FGF acidic, 5 ng/mL rh EGF)

6 mL of L-Alanyl-L-Glutamine (2.4 mM, final concentration)

1 mL of 100 mg/mL G418 (0.2 mg/mL, final concentration)

Subculturing

Protocol: 

1. Passage immortalized adipose-derived MSCs when the culture has reached approximately 80% confluence.

 2.  Warm both the Trypsin-EDTA for Primary Cells (ATCC PCS-999-003) and the Trypsin Neutralizing Solution (ATCC PCS-999-004) to room temperature prior to dissociation. Warm the complete growth medium to 37°C prior to use with the cells.

 3. For each flask, carefully aspirate the spent media without disturbing the monolayer.

 4. Rinse the cell layer one time with 3 to 5 mL D-PBS (ATCC 30-2200) to remove residual medium.

 5. Add prewarmed trypsin-EDTA solution (1 to 2 mL for every 25 cm2) to each flask.

 6. Gently rock each flask to ensure complete coverage of the trypsin-EDTA solution over the cells, and then aspirate the excess fluid off of the monolayer.

 7. Observe the cells under the microscope. When the cells pull away from each other and round up (typically within 3 to 5 minutes), remove the flask from the microscope and gently tap it from several sides to promote detachment of the cells from the flask surface.

 8. When the majority of cells appear to have detached, quickly add an equal volume of the Trypsin Neutralizing Solution (ATCC PCS-999-004) to each flask. Gently pipette or swirl the culture to ensure all of the trypsin-EDTA solution has been neutralized.

 9. Transfer the dissociated cells to a sterile centrifuge tube and set aside while processing any remaining cells in the culture flask.

 10. Add 3 to 5 mL D-PBS (ATCC 30-2200) to the tissue culture flask to collect any additional cells that might have been left behind.

 11. Transfer the cell/D-PBS suspension to the centrifuge tube containing the trypsin-EDTA- dissociated cells.

 12. Repeat steps 10 and 11 as needed until all cells have been collected from the flask.

 13. Centrifuge the cells at 270 x g for 5 minutes.

 14. Aspirate neutralized dissociation solution from the cell pellet and resuspend the cells in 2 to 8 mL fresh, prewarmed, complete growth medium.

 15. Count the cells and seed new culture flasks at a density of 5,000 viable cells per cm2.

 16. Place newly seeded flasks in a 37°C, 5% CO2 incubator for at least 24 to 48 hours before processing the cells further. 

Cell seeding density: 5,000 viable cells per cm2
Medium renewal: every 2 to 3 days
Cryopreservation
Freeze medium: 90% Complete growth media; DMSO, 10%
Storage temperature: liquid nitrogen vapor phase
Culture Conditions
Temperature: 37.0°C
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
STR Profile
Amelogenin: X
CSF1PO: 10, 13
D13S317: 8, 12
D16S539: 10, 13
D5S818: 11, 13
D7S820: 8, 11
THO1: 7
TPOX: 8
vWA: 16
Population Doubling Level (PDL)

Longevity: >25 PDLs post-thaw

Population Doubling Time approximately 45 hours
Name of Depositor Regina Grillari-Voglauer
Year of Origin December 2006
References

Bodnar AG, et al. Extension of life-span by introduction of telomerase into normal human cells. Science 279: 349-352, 1998. PubMed: 9454332

Wolbank S, et al. Telomerase immortalized human amnion- and adipose-derived mesenchymal stem cells: Maintenance of differentiation and immunomodulatory characteristics. Tissue Eng. Part A 15(7): 1843-1854, 2009. PubMed: 19125642

Basic Documentation
Other Documentation
FAQ's
  1. Applications for hTERT-MSCs
    The hTERT immortalized adipose-derived MSCs (ATCC® SCRC-4000 ™) can be differentiated into adipocytes, osteoblasts and chondrocytes.  This makes hTERT-MSCs ideal
    Date Updated: 2/14/2014
  2. Differentiation Potential of hTERT immortalized MSCs
    ASC52telo hTERT-MSCs (ATCC® SCRC-4000 ™) and primary adipose-derived MSCs (ATCC® PCS-500-011 ™) have a similar adipogenesis potential.  The hTERT-MSC culture and the...
    Date Updated: 2/13/2014
  3. Longevity of ASC52telo MSCs
    Based on ongoing longevity studies, ATCC® SCRC-4000 ™ ASC52telo hTERT-MSCs can be grown for at least 22 passages with a normal cell morphology and growth rate when using the ATCC r...
    Date Updated: 2/13/2014
  4. Primary vs. immortalized MSC marker expression

    We have not seen significant differences in MSC marker expression in the ATCC® SCRC-4000 ™ ASC52telo hTERT immortalized MSCs compared to primary MSCs.


    Date Updated: 2/13/2014
  5. MSC differentiation when subconfluent
    No. Adipogenic or osteogenic differenciation is less efficient when the MSCs are subconfluent.  Start adding the adipogenic or osteogenic differentiation medium when the cells reach abo...
    Date Updated: 2/14/2014
  6. Adipogenic medium exchange for hTERT-MSCs
    It is very important that the cell monolayer is not exposed to air during the adipogenic differentiation process so that the lipid vescicles do not burst.  Therefore, do not exchange 100% of...
    Date Updated: 2/13/2014
  7. Source of hTERT MSC culture
    The ASC52telo hTERT immortalized normal human adipose-derived mesenchymal stem cells (ATCC® SCRC-4000 ™) were originally isolated from subcutaneous adipose tissue obtained from out...
    Date Updated: 2/13/2014
Restrictions

This material is subject to claims under U.S. Patent Nos. 6,261,836 and 6,337,200, other pending patent applications, and foreign counterparts thereof. It is required that either the Addendum for Commercial and For-Profit Organizations or the Addendum for Noncommercial and Academic Organizations, as appropriate, be signed and returned to ATCC before shipment.

References

Bodnar AG, et al. Extension of life-span by introduction of telomerase into normal human cells. Science 279: 349-352, 1998. PubMed: 9454332

Wolbank S, et al. Telomerase immortalized human amnion- and adipose-derived mesenchymal stem cells: Maintenance of differentiation and immunomodulatory characteristics. Tissue Eng. Part A 15(7): 1843-1854, 2009. PubMed: 19125642