Immortalized Human Primary Colonic Fibroblasts

$3,000.00

Immortalized Human Primary Colonic Fibroblasts are specialized stromal cells derived from human colon tissue and genetically modified for extended in vitro proliferation while maintaining key physiological properties. These cells exhibit characteristic fibroblast morphology and express standard markers including vimentin, collagen I/III, and fibroblast activation protein (FAP), while testing negative for epithelial (cytokeratin 20) and endothelial (CD31) contaminants. Certified free of mycoplasma, bacteria, yeast, and fungi, the fibroblasts demonstrate stable growth for 25+ passages in optimized fibroblast medium when cultured at 37°C/5% CO₂. Particularly valuable for gastrointestinal research, they are ideal for studying intestinal fibrosis, inflammatory bowel disease mechanisms, tumor microenvironment interactions, and epithelial-mesenchymal crosstalk. Supplied cryopreserved at passage 3 with ≥1×10⁶ viable cells/vial, these colonic fibroblasts retain responsiveness to TGF-β and Wnt signaling pathways and show consistent doubling times of 24-36 hours during logarithmic growth phase. Their robust proliferative capacity and maintained phenotypic stability make them excellent for both short-term functional assays and long-term co-culture studies modeling colonic pathophysiology.

Immortalized Human Primary Colonic Fibroblasts are specialized stromal cells derived from human colon tissue and genetically modified for extended in vitro proliferation while maintaining key physiological properties. These cells exhibit characteristic fibroblast morphology and express standard markers including vimentin, collagen I/III, and fibroblast activation protein (FAP), while testing negative for epithelial (cytokeratin 20) and endothelial (CD31) contaminants. Certified free of mycoplasma, bacteria, yeast, and fungi, the fibroblasts demonstrate stable growth for 25+ passages in optimized fibroblast medium when cultured at 37°C/5% CO₂. Particularly valuable for gastrointestinal research, they are ideal for studying intestinal fibrosis, inflammatory bowel disease mechanisms, tumor microenvironment interactions, and epithelial-mesenchymal crosstalk. Supplied cryopreserved at passage 3 with ≥1×10⁶ viable cells/vial, these colonic fibroblasts retain responsiveness to TGF-β and Wnt signaling pathways and show consistent doubling times of 24-36 hours during logarithmic growth phase. Their robust proliferative capacity and maintained phenotypic stability make them excellent for both short-term functional assays and long-term co-culture studies modeling colonic pathophysiology.


Protocol for Culturing Immortalized Human Primary Colonic Fibroblasts

Materials Required:

  • Immortalized Human Primary Colonic Fibroblasts (Catalog #ICF-001)

  • Complete Colonic Fibroblast Growth Medium (ICFGM001)

  • Sterile 1X PBS (Ca²⁺/Mg²⁺-free, PBS001)

  • 0.25% Trypsin-EDTA Cell Detachment Solution

  • Trypsin Neutralization Solution

  • AlphaBioCoat-coated T25 flasks (CBT-25)

  • 37°C humidified incubator with 5% CO₂

  • Benchtop centrifuge (200-300 × g capacity)

  • Automated cell counter or hemocytometer

  • DMSO-containing cryopreservation medium (CRYO-10)

  • Liquid nitrogen storage tank with vapor phase capability

Detailed Protocol:

1. Cell Thawing and Initial Plating:

  1. Prepare complete growth medium by warming ICFGM001 to 37°C in a water bath.

  2. Rapidly thaw frozen vial in 37°C water bath for 60-90 seconds until only a small ice crystal remains.

  3. Decontaminate vial exterior with 70% ethanol and transfer contents to 15 mL conical tube.

  4. Slowly dilute cell suspension by adding 5 mL pre-warmed medium dropwise over 1 minute.

  5. Centrifuge at 200 × g for 5 minutes at room temperature.

  6. Aspirate supernatant and gently resuspend pellet in 5 mL fresh medium.

  7. Seed cells in AlphaBioCoat-coated T25 flask and incubate at 37°C/5% CO₂.

2. Routine Culture Maintenance:

  • Monitor cell morphology daily using phase contrast microscopy (10-20× magnification)

  • Replace medium every 48-72 hours until 80-90% confluency is reached

  • For passaging:
    a) Aspirate spent medium and rinse with 2 mL PBS
    b) Add 1 mL Trypsin-EDTA and incubate at 37°C for 2-3 minutes
    c) Neutralize with 2 mL pre-warmed neutralization solution
    d) Centrifuge cell suspension at 200 × g for 5 minutes
    e) Resuspend in fresh medium and seed at 1:3 to 1:5 split ratio

3. Cryopreservation Protocol:

  1. Harvest cells at 70-80% confluency as described above

  2. Resuspend cell pellet in CRYO-10 medium at 1-2×10⁶ cells/mL

  3. Aliquot 1 mL into pre-labeled cryovials

  4. Place vials in isopropanol freezing container at -80°C overnight

  5. Transfer to liquid nitrogen vapor phase for long-term storage

Quality Assurance Parameters:

  • Expected viability post-thaw: ≥90% (Trypan Blue exclusion)

  • Typical doubling time: 28-32 hours during log phase growth

  • Recommended passage range: P3-P25 for optimal performance

  • Authentication: STR profiled and tested for mycoplasma contamination

Troubleshooting Guide:

  • Poor attachment: Verify coating procedure and check medium pH (7.2-7.4)

  • Slow proliferation: Confirm proper incubation conditions and medium freshness

  • Morphological changes: Limit passage number and avoid over-confluence

  • Contamination: Implement strict aseptic technique and regular mycoplasma testing

Expected Experimental Outcomes:

  • Consistent spindle-shaped morphology through multiple passages

  • Stable expression of colonic fibroblast markers (FAP, α-SMA, vimentin)

  • Responsiveness to TGF-β and inflammatory cytokine stimulation

  • Suitable for co-culture systems, ECM studies, and fibrosis modeling

Note: All procedures should be performed under sterile conditions in a Class II biological safety cabinet. Optimal results are obtained when cells are maintained below passage 25 and medium is replaced every 2-3 days.