Keep on rolling: optimizing human islet transport conditions using a perfused rotary system.

The setup of an islet isolation facility designed along the rules of good manufacturing practice (GMP) is a technically challenging, cost and time intensive process. ( 1) Consequently, several institutions have decided to perform transplantation of islets isolated at another center with an already standing expertise. Such a solution includes the necessity to transport the isolated islets from the isolation to the transplantation facility. In spite of its importance, an ideal solution for the transport of the isolated human islets has still not been established.   Here, we present an islet transport device suited to transport human islet cells under reproducible conditions and minimized cell stress. The transport simulation of the human islets was performed in a transfused "rotary transport system for islets" termed "ROTi." Besides measuring standard metabolic (LDH, lactate, glucose) and physical parameters (pH, dissolved oxygen and temperature), we used five different live stains in combination with real time live confocal microscopy to document islet quality parameters. As live stains we added tetramethylrhodamine methyl ester, cell permeant acetoxymethylester, propidium iodide, annexin-fitc and fluorescent wheat germ agglutinin, and assessed mitochondrial membrane potentials, calcium levels, cell death, apoptosis or cell morphology, respectively. We compared the viability of human islets after 24 h incubation in the ROTi device to an incubation simulating "standard" shipment of islets in 50 ml tubes. All cell viability parameters studied (mitochondrial membrane potentials, calcium content, apoptosis, cell death as well as cell morphology) documented a significantly better cell viability in the ROTi fraction compared with the simulated "standard" shipment fraction. Besides maintaining islet cell viability, the ROTi bears the advantage of a better reproducibility of islet transport conditions.

Introduction of a novel prototype bioartificial liver support system utilizing small human hepatocytes in rotary culture.

The aim of this study was to establish a stand-alone, perfused, rotary cell culture system using small human hepatocytes (SH) for bioartificial liver (BAL) support. SH were grown on cytodex 3 microcarriers (beads) to a maximum density of 1.2 +/- 0.3 x 10(7) cells per mL within 12 days. Size of aggregates formed by up to 15 beads was regulated by rotation speed. Cell function was proven by treatment with ammonia and galactose, and metabolism was analyzed. Treatment strategy was comprised of two phases, namely growth phase and treatment phase. Cells were grown for 6 days and subsequently incubated with ammonia or galactose for 2 days, followed by a 2-day regeneration period and another 2-day treatment phase. Consumption of glucose, release of lactate dehydrogenase, formation of lactate, and production of urea and albumin were determined regularly. Mean galactose consumption was 50 microg per 106 cells per hour, ammonia-induced urea formation was 3.6 microg per 106 cells per hour, and albumin production was 110 ng per 106 cells per hour. All metabolic parameters followed a logarithmic trend and were found to be very stable in the second half of the culture period when cells were treated with ammonia or galactose. Dissolved oxygen (%DO), pH, and temperature were monitored in-stream at intervals of 7 min, and the values were logged. Viability and morphology of cells were monitored via confocal microscopy. Viability was around 95% in controls and 90% during treatment. Promising results were obtained in support of our ongoing efforts to establish a fully autonomous BAL support device utilizing SH as a bridge to transplantation.

Development of a novel perfused rotary cell culture system.

A rotary cell culture system has been established. System quality was determined by observing the stability of the basic parameters of temperature, gas exchange, and pH, and mass transfer (time to equimolarity) between the medium circuit and the 2 cell-containing chambers was investigated. Mass transfer time for urea and several ions was approximately 30 min for the high-fiber-density chamber (HFC) and 50 min for the low-fiber-density chamber (LFC). Exchange of albumin was delayed in both chambers, highlighting the dependence of mass transfer on area of exchange and molecule size. Finally, the ability for cell growth and maintenance was tested. Densities of up to 1.2 x 10(7) immortalized cells per mL at a viability of up to 85% were obtained after 1 week of continuous, non-interfering culture of immortalized cells in the HFC. Human pancreatic islets were also cultivated in the LFC. Confocal analysis using fluorescent dyes showed that the 3-dimensional islet structure was maintained for 1 week. Promising results were obtained, which will further our ongoing efforts toward establishing a mobile cell culture system.