hTERT Lung Fibroblast

Cells were immortalized via transfection with the catalytic subunit of the human telomerase (hTERT) gene.

In development the following functionality was confirmed:

Positive for TGF-Beta induced Alpha-smooth muscle actin expression. Cells were serum starved for 24 hours before being exposed to TFG-beta for 1 hour. Cells were then fixed with cold methanol and stained for alpha smooth muscle actin (A-SMA) expression.

Chlorhexidine (CHX) has been shown to be somewhat toxic to cultured fibroblasts. Our data show a similar toxicity in hTERT lung fibroblasts compared to primary lung fibroblasts, suggesting that hTERT lung fibroblasts can be an excellent model for investigating toxicity. Primary and hTERT-immortalized lung fibroblast cells were seeded at identical numbers in a 96 well plate. The effect on cell viability of two concentrations of CHX was tested at three time points (1, 2, 3 hours) post-addition of CHX. Untreated cells were used as a negative control. One hour prior to the each time point Reliablue Reagent (ATCC 30-1014) was added to wells and allowed to develop; absorbance was then measured after an hour. Absorbance of treated cells was then compared to negative control cells to determine the percentage of live cells. All samples were done in triplicate.

Conclusion: the hTERT-immortalized lung fibroblast primary cell behaves similarly with its primary counterparts

1. Check all containers for leakage or breakage.
2. Remove the frozen cells from the dry ice packaging and immediately place the cells at a temperature below ­-130°C, preferably in liquid nitrogen vapor, until ready for use.

To ensure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt. If upon arrival, continued storage of the frozen culture is necessary, it should be stored in liquid nitrogen vapor phase and not at -70°C.  Storage at -70°C will result in loss of viability.  

1. Thaw the vial by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water.  Thawing should be rapid (approximately 2 minutes).
2. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by dipping in or spraying with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions.
3. Transfer the vial contents to a centrifuge tube containing  9.0 mL complete culture medium. and spin at approximately 220 x g for 5 to 7 minutes.
4. Resuspend cell pellet with the recommended complete medium (see the specific batch information for the culture recommended dilution ratio). It is important to avoid excessive alkalinity of the medium during recovery of the cells.  It is suggested that, prior to the addition of the vial contents, the culture vessel containing the complete growth medium be placed into the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6). pH (7.0 to 7.6).
5. Incubate the culture at 37°C in a suitable incubator.  A 5% CO₂ in air atmosphere is recommended if using the medium described on this product sheet.

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with D-PBS (ATCC 30-2200) to remove all traces of serum that contains trypsin inhibitor.
3. Add 2.0 to 3.0 mL of Trypsin-EDTA for Primary Cells solution (ATCC PCS-999-003) 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 5 x 10³ and 8 x 10³ viable cells/cm².
6. Incubate cultures at 37°C.

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