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

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

482 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)

2 mL of 50 mg/mL G418 (0.2 mg/mL, final concentration)

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 275 x g +/- 125 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. 

Longevity: >25 PDLs post-thaw

Aprile M, et al. PPARγD5, a naturally occurring dominant-negative splice isoform, impairs PPARγ function and adipocyte differentiation. Cell Rep 25(6) 1577-1592, 2018. PubMed: 30404011

Aprile M, et al. In vitro-generated hypertrophic-like adipocytes displaying PPARγ isoforms unbalance recapitulate adipocyte dysfunctions in vivo. Cells 9(5): 1284, 2020. PubMed: 32455814

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

Farhang N, et al. CRISPR-Based Epigenome Editing of Cytokine Receptors for the Promotion of Cell Survival and Tissue Deposition in Inflammatory Environments. Tissue Eng Part A, 23(15-16):738-749, 2017. PubMed: 28095751

Zitnay JL, et al. Fabrication of dense anisotropic collagen scaffolds using biaxial compression. Acta Biomater 65:76-87, 2018. PubMed: 29128533

Nardone G, et al. YAP regulates cell mechanics by controlling focal adhesion assembly. Nat Commun 8:15321, 2017. PubMed: 28504269

Sim SW, et al. Aberrant proliferation and differentiation of glycogen storage disease type Ib mesenchymal stem cells. FEBS Lett 592(2): 162-171, 2018. PubMed: 29238966

Pitrone M, et al. NANOG Plays a Hierarchical Role in the Transcription Network Regulating the Pluripotency and Plasticity of Adipose Tissue-Derived Stem Cells. Int J Mol Sci 18(6): E1107, 2017. PubMed: 28545230

Aprile M, et al. PPARγD5, a naturally occurring dominant-negative splice isoform, impairs PPARγ function and adipocyte differentiation. Cell Rep 25(6) 1577-1592, 2018. PubMed: 30404011

Aprile M, et al. In vitro-generated hypertrophic-like adipocytes displaying PPARγ isoforms unbalance recapitulate adipocyte dysfunctions in vivo. Cells 9(5): 1284, 2020. PubMed: 32455814

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

Farhang N, et al. CRISPR-Based Epigenome Editing of Cytokine Receptors for the Promotion of Cell Survival and Tissue Deposition in Inflammatory Environments. Tissue Eng Part A, 23(15-16):738-749, 2017. PubMed: 28095751

Zitnay JL, et al. Fabrication of dense anisotropic collagen scaffolds using biaxial compression. Acta Biomater 65:76-87, 2018. PubMed: 29128533

Nardone G, et al. YAP regulates cell mechanics by controlling focal adhesion assembly. Nat Commun 8:15321, 2017. PubMed: 28504269

Sim SW, et al. Aberrant proliferation and differentiation of glycogen storage disease type Ib mesenchymal stem cells. FEBS Lett 592(2): 162-171, 2018. PubMed: 29238966

Pitrone M, et al. NANOG Plays a Hierarchical Role in the Transcription Network Regulating the Pluripotency and Plasticity of Adipose Tissue-Derived Stem Cells. Int J Mol Sci 18(6): E1107, 2017. PubMed: 28545230