PRISM: A Fast, Compact, In-line, High Yield, Human Pancreatic Islet Isolation Method
J.B. Doppenberg, M.A. Engelse, E.J.P de Koning
Chair(s): Dr. B.G. Hepkema
Thursday 9 march 2017
12:30 - 12:45h
Categories: Poster - Basaal
Parallel session: Postersessie - Basaal 1
Islet isolations are technically complex, lengthy, expensive, and require a high expertise level from its operators. A low islet yield and/or poor islet quality that is frequently observed result in a high risk that the isolated islets will not be transplanted.
A new, closed method of tissue collection, washing, buffer change and islet purification termed PRISM (PancReatic Islet Separation Method) was developed. Briefly, digested pancreatic tissue is pumped (200 ml/min) through a Medtronic Biotherm heat exchanger, cooling the tissue to 4°C. Mixed with human serum (6 ml/min), the tissue is then continuously concentrated and washed in an air cooled 225 ml Latham centrifugation bowl at 1400 RPM, kept at 4°C. After increasing centrifugation speed to 1500 RPM, UW solution is pumped (30 ml/min) into the bowl while tissue is retained in the bowl. Next, the UW solution containing the digested tissue is transferred from the bowl into a transfusion bag. Two speed controlled Masterflex pumps subsequently mix lopromide 370 mg I/ml (density 1.409 g/ml) with the digest (suspended in UW) into the bowl until the heaviest tissue has exited the bowl. Microspheres of a similar size, shape and density as digested pancreas tissue, as well as islet depleted tissue (IDP) were used in initial testing of the system. Ten human pancreata, not suitable for clinical use, were used for islet isolation using PRISM. Yield (IEQ), viability (FDA/PI staining) and function (dynamic glucose stimulated insulin secretion test, dGSIS) were evaluated after one day of culture.
Pure fractions of microspheres could be separated in isopycnic centrifugation using the bowl. Microspheres could be concentrated and retained in the bowl without loss at 1400 RPM and pump speed at 200 ml/min. IDP could also be retained in this fashion. Merely one operator can perform PRISM in one flow cabinet. Using this procedure with human pancreata resulted in a higher islet yield when compared to historical controls (431,234±292,833 vs. 285,276±197,392 IEQ, p=0.05). PRISM islets were as functional as historical controls in dGSIS tests (stimulation index of 4.5 vs 4.6 and AUC 351.1 vs. 398.7, respectively; p=ns). Islet viability was 86%. Isolated islets were of similar diameter and shape as historical controls.
PRISM is a novel islet isolation technique that represents a significant improvement in islet isolation efficiency.
