Novel primary culture method of pancreatic duct epithelial cells of rat
A technique for pancreatic ducts and epithelial cells, which is applied in the field of cell biology and biotechnology, can solve the problems of contamination of remaining cells, insufficient digestion, and unsuitability for large-scale production, and achieves the effect of simplifying experimental steps and reducing opportunities for contamination.
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Embodiment 1
[0056] Embodiment 1 Rat pancreatic ductal epithelial cell primary culture method of the present invention
[0057] 1. Main experimental materials
[0058] 1) High serum medium: IMEMZO medium added: 20% fetal bovine serum FBS, 5 μg / ml insulin, 100 ng / ml epidermal growth factor, 4 μg / ml dexamethasone, 100 ng / ml cholera toxin, 100 ng / ml transferrin , 500U / ml penicillin, 5μg / ml streptomycin.
[0059] 2) Low serum medium: 2% fetal bovine serum FBS, 200 U / ml penicillin, and 2 μg / ml streptomycin were added to the IMEMZO medium. The rest of the additives are the same as above.
[0060] 3) Mixed digestive enzymes: 0.1% type V collagenase, 0.1% hyaluronidase, 0.1% soybean trypsin inhibitor. All enzymes were obtained from sigma, St. Louis, Missouri, USA;
[0061] 4) Buffered saline solution (HBSS): add 500 U / ml penicillin, 5 μg / ml streptomycin in Hank's solution.
[0062] The buffered saline solution (HBSS) can be prepared by oneself, or can be directly purchased from a commercial p...
Embodiment 2
[0097] Example 2 Concentration selection test of mixed digestive enzymes in the primary culture method of rat pancreatic ductal epithelial cells of the present invention:
[0098] The operation was carried out according to the method of Example 1, and the concentration of each of the three enzymes was consistent, and four experimental groups of 0.05%, 0.1%, 0.2%, and 0.5% were used for comparison.
[0099] Test results: 0.05% group due to low concentration, many times of digestion and long time, the cells could not fully dissociate from the tissue block. 0.5% has obvious overdigestion during the digestion process, with many cell fragments and gelatinous substances entangled together. The digestion process of 0.1% and 0.2% groups was similar. But from the point of view of the final output, the 0.1% group is more than the 0.2% group, the 0.05% group is more than the 0.5% group. Therefore, the enzyme concentration can be between 0.1% and 0.2%, with 0.1% being optimal.
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