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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.

Inactive Publication Date: 2011-01-26
WEST CHINA HOSPITAL SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

Hou Min, et al., Primary and Subculture of Human Pancreatic Ductal Epithelial Cells, "Acta Anatomy", Volume 31, Issue 2, 2000 The whole pancreas was taken and digested with mixed digestive enzymes. The digestive juice was Hanks equilibrium solution with collagenase IA Type (Sigma) 0.5g / L, soybean trypsin inhibitor (Sigma) makes its final concentration 0.01%. Although the method reported in this literature uses mixed digestive enzymes, the digestion process is not segmented, resulting in some overdigestion, And some are not fully digested
In addition, other reported methods mostly use the whole pancreas for digestion without segmentation, or use different enzymes for different steps
Incorporation of the whole pancreas will greatly increase the contamination of other types of cells, especially fibroblasts; without step-by-step digestion, some cells have not been digested, and some cells have been fragmented due to excessive digestion, reducing cell yield
The Ficoll density gradient centrifugation method is expensive and not suitable for large-scale production; the tissue block culture method takes a long time and the remaining cells are seriously polluted
Technical problems such as how to accurately obtain experimental materials, how to moderately control enzyme digestion steps, and how to effectively remove fibroblast contamination are still controversial.

Method used

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  • Novel primary culture method of pancreatic duct epithelial cells of rat
  • Novel primary culture method of pancreatic duct epithelial cells of rat
  • Novel primary culture method of pancreatic duct epithelial cells of rat

Examples

Experimental program
Comparison scheme
Effect test

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.

[0100]...

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Abstract

The invention provides a novel primary culture method of pancreatic duct epithelial cells of a rat. The method comprises the following steps of: a, separating a main pancreatic duct from a pancreatic tissue of a healthy rat; b, digesting and separating pancreatic tissue fragments by using mixed digestive enzyme by a step-by-step digestion method to obtain cell aggregates; and c, culturing and purifying the cell aggregates, namely rotating a vessel by using a time difference and jointly purifying by using a gradient serum culture medium to obtain the pancreatic duct epithelial cells. In the method, the raw material is taken from the common laboratory animal, namely the rat, and the main pancreatic duct can be quickly and efficiently stripped from the whole pancreatic tissue by using the tension of surrounding organs of the main pancreatic duct. The cell aggregates with a proper size can be obtained by the step-by-step digestion method; and the yield of the cultured cells can be improved by over 60 percent than that of the cultured cells by an overall digestion method. By combining three methods, namely, material taking, time difference-based vessel rotation and the use of the gradient serum culture medium, the pollution of other pancreatic cells is removed, and the epithelial cells are purified, wherein the purification rate of the epithelial cells of the invention can reach 95 percent.

Description

technical field [0001] The invention relates to a new method for primary culture of rat pancreatic ductal epithelial cells, which belongs to the field of cell biology and biotechnology. Background technique [0002] Pancreatic duct epithelial cells (PDEC) are closely related to most common pancreatic diseases, such as pancreatic cancer, pancreatitis, and diabetes. According to reports, more than 90% of pancreatic cancers originate from pancreatic ductal epithelial cells, and in the initial stage of acute pancreatitis, pancreatic ductal epithelial cells not only play the role of mucus barrier, but also secrete high-concentration HCO 3 - The lye inhibits the activity of trypsin to prevent the pancreas from digesting itself. In recent years, hot research has focused on inducing the differentiation of autologous pancreatic ductal epithelial cells into islet cells, which is expected to solve the problems of few sources of transplantable islets and rejection, and its potential v...

Claims

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Application Information

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IPC IPC(8): C12N5/071
Inventor 周总光陈珂玲李园郑雪莲
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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