Sharon C. Presnell

The use of the BD oxygen biosensor system to assess isolated human islets of langerhans: oxygen consumption as a potential measure of islet potency.

The measurement of cellular oxygen consumption rate (OCR) is a potential tool for the assessment of metabolic potency of isolated islets of Langerhans prior to clinical transplantation. We used a commercially available 96-well plate fluoroprobe, the BD Oxygen Biosensor System (OBS), to estimate OCR in 27 human islet preparations, and compared these results to those of concurrent mouse transplantations. OCR was estimated both from the dO2 at steady state and from the transient rate of change of dO2 during the initial culture period immediately after seeding (“dO2 slope”). To demonstrate the validity of the OBS-derived values, it was shown that they scaled linearly with islet equivalent number/DNA concentration and with each other. These measurements were obtained for each preparation of islets incubated in media supplemented with either low (2.2 mM) or high (22 mM) glucose. Concurrently, one to three athymic nude mice were transplanted with 2,000 IEQs under the kidney capsule. The OCR Index, defined as the ratio of the DNA-normalized “dO2 slope” in high glucose to that in low glucose, proved highly predictive of mouse transplant results. Of the 69 mice transplanted, those receiving islets where the OCR Index exceeded 1.27 were 90% likely to reverse within 3 days, whereas those receiving islets with an OCR Index below 1.27 took significantly longer, often failing to reverse at all over a 35-day time period. These results suggest that the OBS could be a useful tool for the pretransplant assessment of islet cell potency.

Nucleic Acid Blotting Techniques

This chapter deals with basic concepts and techniques in nucleic acid blotting. Many of the techniques involved with Southern blotting and Northern blotting are similar. Negatively charged, purified nucleic acids from prokaryotic or eukaryotic cells are separated according to size by electrophoresis through an agarose gel matrix. The RNA or denatured DNA is subse– quently transferred and immobilized onto a membrane com– posed of nitrocellulose or nylon. The nucleic acids on the membrane are then hybridized to a specific labeled “probe,” which consists of homologous single-stranded nucleic acids that carry molecules, allowing detection and visualization of the hybridized probe. Hybridization between the immobilized nucleic acids and labeled probe allows detection of specific DNA or RNA sequences within a complex mixture of DNA or RNA. The specific method of detection and visualization is dependent on the nature of the labeled probe; radioactive probes enable autoradiographic detection, and probes labeled with enzymes facilitate chemiluminescent or colorimetric detection. Nucleic acid blotting yields valuable information pertaining to gene integrity and copy number (Southern blot) and provides a means of analyzing gene expression and mRNA size (Northern blot). These methods can be used to character– ize tissues and cultured cells in the laboratory and often provide valuable information for clinical evaluation of patient samples.

Rapid method for assessing oxygen consumption rate of cells from transient-state measurements of pericellular dissolved oxygen concentration

Recently we described a method for estimating the oxygen consumption rate (OCR) of cells in static culture from equilibrium measurements of dissolved oxygen concentration (dO2), using an oxygen-sensing microplate and the steady-state solution to Ficks Law (Guarino et al. 2004). Here we describe a complementary method for estimating OCR from the transient-state rate of change of measured dO2. Although the system is open to the atmosphere and subject to a significant lag in sensor response, the rate of change of the measured dO2 immediately after seeding correlated directly with both cell number and steady-state OCR. This transient-state method is linear with cell number to a much higher density than is possible with the steady-state method because it derives from measurements made before diffusion limitations can be established. For a given sensor thickness, the same correlation line between the transient and non-diffusion-limited steady-state estimates of OCR was found to apply for various preparations of rat hepatocytes. The correlation slope varied predictably with sensor thickness. Thus, despite the non-idealities of this system, the initial rate measurement offers a rapid method to obtain an estimate of absolute OCR. To demonstrate the utility of this method, we purposefully treated rat hepatocytes in ways expected to change OCR. Cells deprived of oxygen by storage under several centimeters of medium showed decreases in both OCR and viability with time. Likewise, the OCR of hepatocytes exposed to the oxidative phosphorylation inhibitor rotenone decreased, whereas those exposed to the uncoupler dinoseb increased.