A Primary Research On Isolation And Culture Of Rat Pancreatic β-cells

Objectives To search for a method available for isolation and purification of pancreaticβ-cells and explore the most suitable conditions for the culture of pancreaticβ-cells in vitro.Method Normal feeding Wistar rats (about 7 to 8 months old, weigh 250 to 300 gram) were used for this study. In order to get the islet of Langerhans, collagenase V was injected though the common bile duct into the pancreas to digest it. Islets were purified primarily by mesh sieve. To evaluate the activity of the islets, they were dyed with dithizone and stimulated by different concentration of glucose. Then they were put into RPMI-1640 nutritive medium for culture overnight. To obtain the suspension containing single pancreatic cells, the islet should be digested again with trypsin and DNase after washing and sedimentation. The cells were separated and purified in a fluorescence-activated cell sorter (BD FACSAria) in isolation medium containing 2.8mmol/L glucose with an argon laser with 100mW at 488nm.The cells emitted fluorescence between 510nm and 550nm were isolated. The purifiedβ-cells were identified and evaluated by immunohistochemistry and glucose stimulating test. To explore the most appropriate condition for the culture of pancreaticβ-cells in vitro and evaluate whether IBMX have special contribution to them, Ham's F-10 with different concentration of glucose and IBMX were used as nutritive medium.Result: After digesting by collagenase and filtering by mesh sieve of each pancreas, 550±90 islets were obtained. We got 5688β-cells from each rat with recovery of 93.69% and purity of 85.5%. Separatedβ-cells were cultured in Ham's F-10 containing different concentration of glucose and IBMX. Relatively high survival rate was observed when the concentration of glucose was more than 10mmol/L. Unfortunately, the contribution of IBMX toβ-cell's activity and survival rate didn't occur.Conclusion: 1. When the pancreatic islet isolated from normal feeding healthy rat were digested into single cell suspension by trypsin and DNase, FACS could be employed to purify pancreaticβ-cells from the suspension after the cells were put into the medium containing 2.8mmol/L glucose. But the purity ofβ-cell was not satisfactory in our study.2. When carrying out primary culture, pancreaticβ-cells could maintain relatively high activity in Ham's F-10 medium containing more than 10mmol/L glucose from initial study. We couldn't make the conclusion that certain concentration of IBMX have an impact onβ-cells. Further advanced study is needed to explore the most appropriate condition for pancreaticβ-cells and the methods to increase the survival rate in primary culture.