Immortalized Human Primary Pancreatic Fibroblasts

$3,000.00

Immortalized Human Primary Pancreatic Fibroblasts are specialized stromal cells derived from pancreatic tissue and genetically engineered for extended in vitro proliferation while maintaining essential fibroblast functions. These cells exhibit characteristic spindle-shaped morphology and express standard fibroblast markers including vimentin, fibroblast activation protein (FAP), and collagen I, while testing negative for epithelial (E-cadherin) and endothelial (CD31) lineage markers. Certified free of mycoplasma, bacteria, and fungal contamination, the cells demonstrate stable growth for 25+ passages in optimized fibroblast medium when cultured at 37°C/5% CO₂. Particularly valuable for pancreatic research, they are ideal for studying pancreatic fibrosis, tumor-stroma interactions in pancreatic cancer, and the role of fibroblasts in pancreatitis progression. Supplied cryopreserved at passage 3 with ≥1×10⁶ viable cells/vial, these pancreatic fibroblasts retain responsiveness to TGF-β and PDGF signaling pathways and exhibit consistent doubling times of 24-36 hours during logarithmic growth. Their robust proliferative capacity and maintained ECM production capabilities make them particularly suitable for modeling pancreatic fibrotic microenvironments, drug screening applications, and co-culture studies with pancreatic epithelial or cancer cells.

Immortalized Human Primary Pancreatic Fibroblasts are specialized stromal cells derived from pancreatic tissue and genetically engineered for extended in vitro proliferation while maintaining essential fibroblast functions. These cells exhibit characteristic spindle-shaped morphology and express standard fibroblast markers including vimentin, fibroblast activation protein (FAP), and collagen I, while testing negative for epithelial (E-cadherin) and endothelial (CD31) lineage markers. Certified free of mycoplasma, bacteria, and fungal contamination, the cells demonstrate stable growth for 25+ passages in optimized fibroblast medium when cultured at 37°C/5% CO₂. Particularly valuable for pancreatic research, they are ideal for studying pancreatic fibrosis, tumor-stroma interactions in pancreatic cancer, and the role of fibroblasts in pancreatitis progression. Supplied cryopreserved at passage 3 with ≥1×10⁶ viable cells/vial, these pancreatic fibroblasts retain responsiveness to TGF-β and PDGF signaling pathways and exhibit consistent doubling times of 24-36 hours during logarithmic growth. Their robust proliferative capacity and maintained ECM production capabilities make them particularly suitable for modeling pancreatic fibrotic microenvironments, drug screening applications, and co-culture studies with pancreatic epithelial or cancer cells.

Materials Required

  • Immortalized Human Primary Pancreatic Fibroblasts

  • Complete Immortalized Pancreatic Fibroblast Growth Medium (IPFGM001)

  • Sterile Phosphate-Buffered Saline (1× PBS; PBS001)

  • Cell Detachment Solution

  • Alphabiocoat-Coated Tissue Culture Flasks (T25)

  • 37°C Humidified Incubator with 5% CO₂

  • Centrifuge (for passaging, if necessary)

  • Hemocytometer or Automated Cell Counter

  • Cryopreservation Medium

  • Liquid Nitrogen Storage System (for long-term preservation)

Cell Culture Protocol

1. Thawing Frozen Cells

  • Warm Medium: Pre-warm the complete fibroblast growth medium to 37°C.

  • Thaw Cells: Retrieve cryovial from liquid nitrogen and thaw quickly in a 37°C water bath (approximately 1–2 minutes). Immediately disinfect the vial with 70% ethanol.

  • Transfer: Gently pipette the thawed cell suspension into a 15 mL conical tube. Slowly add 5–10 mL of pre-warmed growth medium dropwise to reduce osmotic shock.

  • Centrifuge: Spin at 200 × g for 5 minutes to pellet the cells.

  • Resuspend & Plate: Carefully aspirate the supernatant, resuspend the pellet in fresh medium, and transfer to a T25 Alphabiocoat-coated flask.

  • Incubate: Place the flask in a 37°C, 5% CO₂ humidified incubator.

2. Routine Maintenance & Passaging

  • Monitor Cell Health: Observe cells daily using a microscope. Healthy fibroblasts should display a spindle-like morphology.

  • Media Change: Replace spent medium every 2–3 days or as needed.

  • Passaging (at ~80–90% Confluence):

    • Aspirate the old medium and gently rinse with 1–2 mL of PBS.

    • Add 1–2 mL of Cell Detachment Solution and incubate for 2–5 minutes at 37°C. Monitor detachment under a microscope.

    • Add 2–3 mL of Neutralizing Solution to stop enzymatic activity.

    • Gently pipette to achieve a single-cell suspension.

    • Optional: Centrifuge at 200 × g for 5 minutes if needed.

    • Resuspend in fresh medium and replate at a 1:2 to 1:4 split ratio depending on growth rate.

3. Cryopreservation

  • Harvest: Detach and collect cells using the standard passaging procedure (steps 2.2–2.5).

  • Count: Determine cell density using a hemocytometer or automated counter.

  • Prepare for Freezing: Resuspend cells in cryopreservation medium at 1–5 × 10⁶ cells/mL.

  • Aliquot: Dispense 1 mL of the suspension into each cryovial.

  • Freeze Gradually: Place cryovials in an isopropanol-based freezing container and store at –80°C overnight.

  • Long-Term Storage: Transfer vials to a liquid nitrogen tank the next day for long-term preservation.

4. Quality Control & Troubleshooting

  • Contamination Check: Routinely inspect cultures for signs of contamination such as turbidity or changes in pH (e.g., yellowing medium).

  • Morphology: Healthy fibroblasts are elongated and spindle-shaped. Rounded cells may indicate stress, overgrowth, or detachment.

  • Doubling Time: Immortalized fibroblasts should have a consistent doubling time of ~24–48 hours.

Expected Results

  • Cells should adhere to the flask surface within 24 hours of seeding.

  • Proliferation typically begins within 24–48 hours post-thaw or passage.

  • Immortalized pancreatic fibroblasts can generally be passaged 20–30 times before showing signs of senescence (if not fully immortalized).