Hypothermic oxygenated machine perfusion of the human pancreas for clinical islet isolation: a prospective feasibility study

Abstract

Due to an increasing scarcity of pancreases with optimal donor characteristics, islet isolation centers utilize pancreases from extended criteria donors, such as from donation after circulatory death (DCD) donors, which are particularly susceptible to prolonged cold ischemia time (CIT). We hypothesized that hypothermic machine perfusion (HMP) can safely increase CIT. Five human DCD pancreases were subjected to 6 h of oxygenated HMP. Perfusion parameters, apoptosis, and edema were measured prior to islet isolation. Five human DBD pancreases were evaluated after static cold storage (SCS). Islet viability, and in vitro and in vivo functionality in diabetic mice were analyzed. Islets were isolated from HMP pancreases after 13.4 h [12.9-14.5] CIT and after 9.2 h [6.5-12.5] CIT from SCS pancreases. Histological analysis of the pancreatic tissue showed that HMP did not induce edema nor apoptosis. Islets maintained >90% viable during culture, and an appropriate in vitro and in vivo function in mice was demonstrated after HMP. The current study design does not permit to demonstrate that oxygenated HMP allows for cold ischemia extension; however, the successful isolation of functional islets from discarded human DCD pancreases after performing 6 h of oxygenated HMP indicates that oxygenated HMP may be a useful technology for better preservation of pancreases.

Keywords: donation after circulatory death; hypothermic machine perfusion; islet isolation.

Figure 1

Transportable dual arterial oxygenated hypothermic machine perfusion device. The perfusion system consists of two separate centrifugal pumps (1), two hollow fiber oxygenators (2), perfusion pressure, flow and temperature sensors (3), an organ holder (4) in an insulated container filled with melting ice (5).

Figure 2

Average flow rates through the splenic artery and superior mesenteric artery during HMP. Perfusion flow in both arteries stabilized after 30 min of perfusion and remained so during 6 h of machine perfusion. Flow in the SMA was higher than in the SA in all DCD pancreases. SA, splenic artery; SMA, superior mesenteric artery.

Figure 3

Macroscopic aspect of the pancreas directly after hypothermic machine perfusion. No visible indication of edema formation after machine perfusion was seen.

Figure 4

Change in percentage of edema in HMP and SCS pancreases. Edema was measured as a percentage of interstitial space divided by the total area of tissue measured after H&E staining. The difference in percentage after and before either HMP or SCS is shown. HMP, hypothermic machine perfusion; SCS, static cold storage. Results are shown as box‐and‐whisker median ± interquartile range.

Figure 5

Islet isolation parameters. (a) Percentage of digested pancreas tissue [g/g]. (b) Maximum islet purity after density separation. (c) Average purity of combined islet fractions (≥25% purity). (d) Percentage of embedded islets as part of all isolated islets. HMP, hypothermic machine perfusion; SCS, static cold storage. Results are shown as box‐and‐whisker median ± interquartile range.

Figure 6

Changes in total IEQ after islet isolation from SCS and HMP pancreases. IEQ after islet isolation (day 0), after one day of culturing (day 1), and after three days of culturing (day 3). IEQ, islet equivalent; HMP, hypothermic machine perfusion; SCS, static cold storage. Results are shown as box‐and‐whisker median ± interquartile range.

Figure 7

Postisolation viability of SCS and HMP pancreases. Islet viability, as assessed by FDA‐PI staining, was calculated directly after isolation (day 0), after the first medium change (day 1), and after the second medium change (day 3). HMP, hypothermic machine perfusion; SCS, static cold storage. Results are shown as box‐and‐whisker median ± interquartile range.

Figure 8

Average glucose‐stimulated insulin response in islets from SCS and HMP pancreases. After day 1 medium change, cultured islets from every isolation were perfused in duplo with a low concentration glucose solution (1.7 mM), followed by a high concentration glucose solution (20 mM, gray area), and again by the low concentration glucose solution. Each time point is averaged per group, shown as mean ± SD.

Figure 9

In vivo functionality of islets isolated from SCS and HMP pancreases. In vivo functionality of the isolated islets from HMP‐ and SCS‐preserved pancreases as assessed via transplantation of islets under the kidney capsule of diabetic mice three days after islet isolation. (a) Fold increase in insulin concentrations during the IPGTT at day 28. Insulin concentrations at each time point were divided by insulin concentrations at t = 120 min, averaged over all mice. (b) Glucose concentrations during the intraperitoneal glucose tolerance test (IPGTT) at day 28 after transplantation.