hTERT Human Lung Fibroblasts Squamous Cell Carcinoma

$6,000.00

General Information:
Puromycin-resistant hTERT-immortalized cells are derived from Human Lung Cancer-Associated Fibroblasts (HLCCAF) following lentiviral transduction with vectors expressing the human telomerase reverse transcriptase (hTERT) gene. These CAFs are originally isolated from human lung tumor tissue and initially cultured in T-75 tissue culture flasks pre-coated with AlphaBioCoat for 30 minutes. Cells are maintained in CAF Growth Medium for 3 to 7 days, allowing for initial attachment and proliferation, after which cultures are expanded. Prior to shipment, cells at passage 1 are harvested, detached, and cryopreserved in cryovials. Each vial contains a minimum of 1,000,000 viable cells per milliliter.

General Information:
Puromycin-resistant hTERT-immortalized cells are derived from Human Lung Cancer-Associated Fibroblasts (HLCCAF) following lentiviral transduction with vectors expressing the human telomerase reverse transcriptase (hTERT) gene. These CAFs are originally isolated from human lung tumor tissue and initially cultured in T-75 tissue culture flasks pre-coated with AlphaBioCoat for 30 minutes. Cells are maintained in CAF Growth Medium for 3 to 7 days, allowing for initial attachment and proliferation, after which cultures are expanded. Prior to shipment, cells at passage 1 are harvested, detached, and cryopreserved in cryovials. Each vial contains a minimum of 1,000,000 viable cells per milliliter.

Product Testing

Human Lung Cancer-Associated Fibroblasts (CAF) are rigorously validated for quality and cell identity. Testing confirms:

  • Negative expression for:

    • von Willebrand Factor (Factor VIII)

    • Cytokeratin 18

    • Alpha-smooth muscle actin

  • Negative for microbial contamination, including:

    • Bacteria

    • Yeast

    • Fungi

    • Mycoplasma

  • Positive expression of CAF markers, including:

    • FAP (Fibroblast Activation Protein)

    • PDGFR (Platelet-Derived Growth Factor Receptor)

    • Vimentin

    • PDPN (Podoplanin)

    • CD70

Cells are expansion-capable for 3–5 passages using a split ratio of 1:2 or 1:3 under standard culture conditions.

Laboratory Applications

Human Lung CAFs are suitable for a broad range of in vitro research applications, including:

  • Cell-cell interaction assays

  • Cell adhesion studies

  • PCR and gene expression profiling

  • Western blotting

  • Immunoprecipitation

  • Flow cytometry and immunofluorescence

  • Generation of cell derivatives for advanced biomedical research

Shipping & Handling – Frozen Vials on Dry Ice

To preserve viability, cells are shipped frozen on dry ice.

Upon Receipt:

  • Thaw and culture immediately for best results.

  • If storage is required, maintain vials in the liquid nitrogen vapor phase.

  • Do not store at -70°C, as this leads to significant viability loss.

Thawing Protocol:

  1. Thaw the vial rapidly (within ~2 minutes) in a 37°C water bath, ensuring the O-ring and cap remain dry to avoid contamination.

  2. Once thawed, sanitize the vial using 70% ethanol, and proceed under aseptic conditions.

  3. Centrifuge at 125 × g for 5–10 minutes to remove the cryoprotectant. Discard the supernatant.

  4. Resuspend the pellet in fresh, pre-warmed complete growth medium.

  5. Pre-coat a T-flask with 6–8 mL of AlphaBioCoat for 15 minutes. Aspirate and rinse with 8 mL of 1X PBS, then discard the rinse.

  6. Transfer cells to the coated flask.

  7. Pre-warm the growth medium in the incubator for 15 minutes prior to cell plating to maintain optimal pH (7.0–7.6).

  8. Incubate at 37°C, in a humidified atmosphere with 5% CO₂.

Subculturing Procedure

Recommended Vessel: T-75 flasks
Note: Adjust volumes proportionally for different culture sizes.

⚠️ Avoid shaking or tapping the flask to detach cells, as this may cause clumping. If needed, incubate at 37°C to assist detachment.

Steps:

  1. Remove and discard spent medium.

  2. Rinse cells with 1X PBS to eliminate residual serum inhibitors.

  3. Add 2–3 mL of Cell Detachment Solution and monitor under an inverted microscope until cells begin to lift (typically 5–15 minutes).

  4. Pre-coat a new T-flask with 6–8 mL of AlphaBioCoat for 15 minutes. Rinse with 8 mL of 1X PBS and discard the rinse.

  5. Resuspend detached cells in 6–8 mL of complete growth medium by gentle pipetting.

  6. Seed appropriate volumes of the cell suspension into new culture vessels.

  7. Incubate at 37°C with 5% CO₂.

  8. Subculturing Ratio: 1:2 to 1:3

  9. Medium Renewal: Every 3–4 days

Cryopreservation

For long-term storage, cryopreserve cells in complete growth medium supplemented with 5% (v/v) DMSO.