Pancreas retrieved from multi-organ donor is surface disinfected
Excess fat and connective tissue is removed from the pancreas surface
The pancreas is carefully connected to the Perfusion pump
Digestive enzymes are pumped into the pancreas using the Perfusion pump
Pancreas is placed inside the Ricordi chamber
Media is heated up and circulated through the Ricordi chamber in this open loop system
Ricordi chamber is shaken to facilitate mechanical and enzymatic digestion of the pancreas
Pancreas is digested into acinar and islets. Islets can be visualized (red) by staining with dithizone
Once enzymatic digestion is completed, the pancreas is reduced to a volume of 50g comprising of acinar and islets
The digested pancreatic tissue is purified using gradients of various densities
Gradient purification process using the COBE 2991 Cell Processor, separates the islets from the acinar cells
The pure islets are then collected and cultured
Dithizone staining helps visualize the islets by staining them red
Viability fluorescent staining identifies dead (red) versus alive (green) islets
Purified islets are cultured in flasks and incubated at constant temperature
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