Ear tissue was collected and minced from newborn piglets to create ear fibroblast cultures from female CMAH KO Minnesota miniature pig genetic background 5 (link). The minced tissue was digested in 20 ml of digestion medium (Dulbecco's modified Eagle's medium containing 2.77 mm glucose, 1.99 mm l -glutamine, 0.5 mm sodium pyruvate (Cellgro, Manassas, VA, USA) supplemented with 200 units/ml collagenase and 25 Kunitz units/ml DNaseI) for 5 h at 38.5 °C. After digestion, ear fibroblast cells were washed and cultured with DMEM, 15% fetal bovine serum (FBS, Cellgro) with 40 μg/ml gentamicin (Cellgro). After overnight culture, cells were trypsinized and frozen at -80 °C in aliquots of FBS with 10% dimethyl sulfide (DMSO, Sigma, St. Louis, MO, USA) and then stored in liquid nitrogen.
Protocol detail
Establishing Porcine Ear Fibroblast Cultures
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Cells
Collagenase
Digestion
Dimethyl sulfide
Dmso
Female
Fibroblast cells
Frozen
Genetic background
Gentamicin
Glucose
Glutamine
Newborn
Nitrogen
Pyruvate
Sodium
Tissue
Corresponding Organization :
Other organizations : University of Missouri, Konkuk University, CHA University, Virginia Tech
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Variable analysis
independent variables
- Ear tissue collection method (mincing)
- Digestion medium (Dulbecco's modified Eagle's medium containing 2.77 mM glucose, 1.99 mM L-glutamine, 0.5 mM sodium pyruvate supplemented with 200 units/ml collagenase and 25 Kunitz units/ml DNaseI)
- Digestion duration (5 h)
- Digestion temperature (38.5 °C)
- Establishment of ear fibroblast cultures from female CMAH KO Minnesota miniature pig genetic background 5
- Genetic background (female CMAH KO Minnesota miniature pig)
- Cell culture medium (DMEM, 15% fetal bovine serum with 40 μg/ml gentamicin)
- Cell freezing (FBS with 10% DMSO, stored in liquid nitrogen)
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