J.B. Doppenberg, M.A. Engelse, E.J.P de Koning
Chair(s): prof. dr. Carla C. Baan, Erasmus MC Rotterdam
Thursday 10 march 2016
12:30 - 13:00h at Foyer
Parallel session: Postersessies XI - Opgesplitst in 3 tijdblokken en 3 categoriëen (klinisch, basaal, donatie)
Currently, islet isolations are expensive, lengthy and require a high expertise level from its operators. Our aim is to increase the efficiency of islet isolation. We present a new, single pass, closed method of tissue collection, washing, buffer change and islet purification. Briefly, digested pancreatic tissue is mixed with human serum (1:22) and pumped (200 ml/min) through a Medtronic Biotherm II heat exchanger, cooling the tissue to 4°C. The tissue is then continuously concentrated and washed in a 225 ml Latham centrifugation bowl at 1400 RPM. After increasing centrifugation speed to 1500 RPM, tissue is retained in the bowl and UW solution is pumped in to change buffer. Next, the contents of the bowl are emptied into a bag. For density separation in the centrifuge bowl , two speed controlled Masterflex pumps are used to mix the digest in UW with an incremental amount of Iopromide (1.32 g/ml). For most of the process only one operator is required. Three research pancreata were used to optimize system parameters and settings (centrifuge speeds, solution amounts, density gradient profile). One human islet isolation was performed in which the digested tissue was equally split between the novel method and the current method. One complete isolation was performed using optimized parameters. Dynamic GSIS was performed one day after culture. During optimization IEQ were promising (104,674, 227,174 and 206,522) despite technical difficulties. Comparing the novel and current method by dividing the digest, only a 13% difference in IEQ was obtained in favour of the current method. Using the optimized parameters, 728,261 IEQ (6089 IEQ/g) was isolated in 1 full isolation according to the novel method. High islet purities and almost islet-free exocrine tissue was obtained. No difference in GSIS stimulation index was found between the two methods. Markedly, total isolation time (after set-up) was under 3 hours. This novel technique, while still being optimized, represents a significant improvement in islet isolation efficiency.