Derivation of primary PDAC cell lines were performed as previously described36 (link). Fresh tumors were minced with sterile razor blades, digested with dispase II (17105041, Gibco, 4 mg/ml)/collagenase IV (17104019, Gibco, 4 mg/ml)/RPMI for 1 h at 37°C, filtered by a 70 μm cell strainer, resuspended in RPMI/20%FBS and then seeded on collagen I coated plates (087747, Fisher Scientific). Cells were maintained in RPMI medium with 20% FBS and 1% penicillin, streptomycin and amphotericin B (PSA) antibiotic mixture. Cancer cells were further purified by FACS based on YFP or E-Cadherin expression (anti-E-cadherin antibody, 50-3249-82, eBioscience, 1:100). The sorted cells, using BD FACSAria™ II sorter (South Campus Flow Cytometry Core Lab of MD Anderson Cancer Center) were subsequently expanded in vitro. All studies were performed on cells cultivated less than 30 passages. As these are primary cell lines no further authentication methods were applicable and no mycoplasma tests were performed.
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Derivation of Primary PDAC Cell Lines
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Amphotericin b
Anti antibody
Antibiotic
Cadherin
Cancer
Cell lines
Cells
Collagen i
Collagenase
Dispase ii
E cadherin
Flow cytometry
Mycoplasma
Pdac
Penicillin
Sterile
Streptomycin
Tumors
Corresponding Organization : Baylor College of Medicine
Other organizations : The University of Texas MD Anderson Cancer Center
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Variable analysis
independent variables
- Digestion of fresh tumors with dispase II (17105041, Gibco, 4 mg/ml)/collagenase IV (17104019, Gibco, 4 mg/ml)/RPMI for 1 h at 37°C
- Filtering the digested tumor cells through a 70 μm cell strainer
- Resuspending the cells in RPMI/20% FBS and seeding them on collagen I coated plates (087747, Fisher Scientific)
- FACS-based purification of cancer cells using YFP or E-Cadherin expression (anti-E-cadherin antibody, 50-3249-82, eBioscience, 1:100)
- Establishment and expansion of primary PDAC cell lines
- RPMI medium with 20% FBS and 1% penicillin, streptomycin and amphotericin B (PSA) antibiotic mixture as the cell culture conditions
- Cell passages (all studies were performed on cells cultivated less than 30 passages)
- No positive or negative controls were explicitly mentioned in the provided information.
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