hTERT A41hWAT-SVF (ATCC® CRL-3386)

Organism: Homo sapiens, human  /  Tissue: superficial neck fat; adipose tissue  / 

Permits and Restrictions

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Organism Homo sapiens, human
Tissue superficial neck fat; adipose tissue
Product Format frozen 1.0 mL
Morphology fibroblast-like
Culture Properties adherent
Biosafety Level 2

Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.

Age 56 years
Gender male
Applications

The differentiation capacity of the cells has been tested using the differentiation protocol described in Xue et al (Nature Medicine 2015). Oil Red O staining showed that the majority of cells were differentiated into lipid-laden adipocytes. Gene expression analysis demonstrated differentiated A41 hBAT expressed high level of adipocyte markers such as PPARg, FABP4, and brown fat-specific marker UCP1.

Storage Conditions liquid nitrogen vapor phase
Images
Comments Primary hWAT-SVF cells were infected with retroviruses expressing the plasmid pBABE-Neo-hTERT (Addgene Plasmid #1774, Cambridge, MA) Following retrovirus infection, cells were selected with 700 µg/ml G418 for two weeks.
Complete Growth Medium The base medium for this cell line is Dulbecco's Modified Eagle's Medium (DMEM; 30-2002). To make the complete medium add the following component to 500 mL base medium:
  • 56 mL Fetal Bovine Serum (FBS; ATCC 30-2020) for a final concentration of 10%
Subculturing
Volumes used in this protocol are for 75 cm2 flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes. Corning® T-75 flasks (catalog #430641) are recommended for subculturing this product.
  1. Remove and discard culture medium.
  2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin- 0.53 mM EDTA solution to remove all traces of serum that contains trypsin inhibitor.
  3. Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
    Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
  4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
  5. Add appropriate aliquots of the cell suspension to new culture vessels.
    Cultures can be established between 1.5 x 104 and 4.0 x 104 viable cells/cm2.
  6. Incubate cultures at 37°C.
Interval: Maintain cultures at a cell concentration between 1.0 X 104 and 2.0 X 105 cell/cm2.
Subcultivation Ratio: A subcultivation ratio of 1:3 to 1:8 is recommended
Medium Renewal: 2 to 3 times per week
Cryopreservation Complete culture medium + 10% DMSO (ATCC 4-X)
Culture Conditions
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
Temperature: 37°C
Cells per Vial Approximately 2.5 to 3.5 x 106 cells
Volume 1.0 mL
STR Profile
Amelogenin: X,Y
CSF1PO: 11,13
D13S317: 12,13
D16S539: 8,11
D5S818: 11,12
D7S820: 10,12
THO1: 6,7
TPOX: 8
vWA: 18,19
Sterility Tests Bacteria and yeast: No growth
Mycoplasma: No growth
Viral Testing Hepatitis B: None detected
Cytomegalovirus: None detected
Human immunodeficiency virus: None detected
Epstein-Barr virus: None detected
Human papillomavirus: None detected
Viability ≥ 50%
Population Doubling Time 48-72 hours
Name of Depositor Joslin Diabetes Center, Inc.
Year of Origin 2012
References

Xue R, et al. Clonal analyses and gene profiling identify genetic biomarkers of the thermogenic potential of human brown and white preadipocytes. Nat Med 21(7):760-768, 2015. PubMed: 26076036

Shamsi F, Tseng YH. Protocols for Generation of Immortalized Human Brown and White Preadipocyte Cell Lines. Methods Mol Biol 1566:77-85, 2017. PubMed: 28244042

Basic Documentation
Other Documentation
Restrictions

For commercial accounts, this cell line is only distributed under the terms of a fully signed and executed ATCC® Material Transfer Agreement and Addendum. If the commercial account is screening per completed Addendum, the recipient will be required to pay a Screening Fee (ATCC® ACS-2103F™).

Screening Use is defined as use of Biological Material in small molecule and biologic drug discovery, including initial target identification and validation, assay development, high throughput screening, hit identification, lead optimization, and selection of candidates for clinical development.

If the commercial account is not screening per the completed Addendum, the recipient will not be required to pay a Screening Fee.

References

Xue R, et al. Clonal analyses and gene profiling identify genetic biomarkers of the thermogenic potential of human brown and white preadipocytes. Nat Med 21(7):760-768, 2015. PubMed: 26076036

Shamsi F, Tseng YH. Protocols for Generation of Immortalized Human Brown and White Preadipocyte Cell Lines. Methods Mol Biol 1566:77-85, 2017. PubMed: 28244042