(5ac) Development of Quantitative Methods to Assess Islets of Langerhands

Introduction Transplantation of isolated islets of Langerhans has promising potential to cure type 1 diabetes by inducing long-term normoglycemia and insulin independence. The feasibility of clinical islet transplantations has been established according to the Edmonton Protocol. However, results are variable, and it is not yet possible to predict transplantation outcome from in vitro measurements with islet preparations. Currently, islet enumeration is based on microscopic visualization after staining with a zinc-specific binding dye (dithizone, DTZ) and manual counting. Islet viability is based on a live/dead staining test with two fluorescent dyes, fluorescein diacetate (FDA) and propidium iodide (PI). These methods are operator-dependent and prone to error due to the islet size and shape and are not predictive of transplantation outcome. We developed quantitative assays that allowed reproducible evaluation of meaningful properties that affect the clinical outcome in impure human islet preparations.

Materials and Methods We examined light microscopic (LM) morphological analysis of 1-μm sections to estimate the islet volume fraction and compared the results with those of electron microscopy (EM) ultrastructural analysis on the same preparations. Samples were processed to produce 1-μm sections in epoxy resin. These sections were analyzed with LM (stereological point counting) to give the volume fraction of islet tissue. For quantifying the total number of cells in a preparation, we developed an assay based on nuclei counting. Islet aliquots were incubated with a lysis consisting citric acid and a surfactant for 5 min. The suspension was then sheared through a hypoderminc needle to liberate the nuclei. Liberated nuclei were counted by three methods: (1) visual counting in a hemacytometer after staining with crystal violet, and automatic counting by (2) aperture resistance measurements (Coulter Multisizer II) and (3) flow cytometer measurements (Guava PCA) after staining with the fluorescent dye 7-aminoactinomycin D. The methods differed in the way nuclei were distinguished from fragments, accuracy, time required and range of linearity. Total amount of tissue was also quantified by DNA measurements with a commercially available DNA kit, using the CyQUANT fluorescent dye. A theoretical framework was developed in order to combine volume fraction data from the LM analysis with the total number of cells in the tissue from nuclei measurements in order to estimate the total number of islets present in impure preparations. For five research human preparations, the number of islets (or islet equivalents) present was estimated by manual counting with dithizone staining and 6-12 hr later by nuclei counting with LM analysis. To evaluate tissue viability, we used oxygen consumption rate measurements (OCR), an assay of mitochondrial function. We developed a very small stirred chamber system (Micro Oxygen Uptake System, Model FO/SYS210T, Instech Laboratories, Plymouth Meeting, PA) specifically designed for measurements with islets. The chamber has a volume of about 200 ?Ýl, is stirred with a tiny, glass-coated magnetic stirring bar, is water-jacketed for temperature control, and contains a fiber-optic oxygen sensor that has a fluorophor gel overlain by silicone rubber at the tip of the fiber. After cells or islets are loaded through the top of the chamber, a beveled glass plug is inserted, any excess fluid is expelled from a port, and the plug is rotated to seal the chamber. If the viability, as reflected by OCR, does not change during the course of the experiment, and the minimum pO₂ in the islet remains far above the Michaelis constant for oxygen consumption, then the slope ΔpO₂/Δt is constant, from which OCR is calculated. OCR measurements combined with an assay of total amount of tissue quantification (nuclei counting or DNA analysis) provide a measure of the tissue fractional viability. Transplantation experiments were performed with rat islets implanted into mice and human high purity islets into mice to test the predictive capability of our assays.

Results LM analysis gave results that were very similar to EM analysis. For the research preparations, the islet fraction estimated by dithizone staining was overestimated on average by 40% compared to the islet volume fraction by LM. For total amount of tissue quantification, nuclei counting by flow cytometry provided accurate, precise and fast results, linear in concentration up to about 5 x 10⁵ nuclei/ml. Data of visual counting were linear with concentration over a wider range but were higher than the other methods by an average of 12% because of the inability to consistently distinguish fragments from true nuclei. Precision improved with increasing counts in a manner consistent with data following a Poisson distribution. Samples with about 125 or more islets and 1000 nuclei counted provided a coefficient of variation of about 6% or less. DNA analysis with CyQUANT dye and nuclei counting yielded average values of 6.1 and 6.4 pg DNA/nuclei for rat and human islet preparations, respectively, which are consistent with literature data. The number of islet equivalents estimated by manual counting and DTZ staining were overestimated compared to that obtained by LM data and nuclei counts by an average of 90%. For the OCR measurements, the precision of the estimate from a single OCR measurement was excellent with a coefficient of variation (COV) of about 2% with 250 viable islet equivalents (VIE) and 1% with 500 VIE. With triplicate samples from the same preparation, the COV is higher because of sampling errors and possible heterogeneity of OCR/cell in different islets. COV is about 10% for 250 VIE and 6% for 500 VIE. From a large number of OCR, nuclei counts, and DNA measurements for rat, porcine, and human islet preparations, the distributions of OCR/nucleus and OCR/DNA were created. Similar measurements were combined with a quantititative membrane integrity test to obtain the OCR/viable cell or OCR/DNA of viable cell. The ratio of OCR/cell over OCR/viable cell is a measure of the fractional viability of the preparation. This ratio was calculated using (OCR/DNA)viable = 480 nmol/min mg DNA, a value comparable to the higher value of OCR/DNA we have measured with islets of any species. Membrane integrity was also measured by live/dead staining using FDA/PI for many (but not all) of the same preparations and expressed as fractional viability. Fractional viability estimated by membrane integrity test FDA/PI was much higher than the one obtained by OCR measurements, demonstrating the insensitivity of membrane integrity measurements as an indicator of fractional viability as currently practiced with islet preparations. The data from our transplantation experiments show that the transplantation outcomes may be segregated in three regions (all cure, some cure, none cure), defined by the knowledge of OCR and OCR/DNA (or OCR/cell).

Conclusions The assays we have developed for tissue evaluation provide useful information on tissue properties and are predictive of transplantation outcome.