Quantitative analysis of cell composition and purity of human pancreatic islet preparations

Abstract

Despite improvements in outcomes for human islet transplantation, characterization of islet preparations remains poorly defined. This study used both light microscopy (LM) and electron microscopy (EM) to characterize 33 islet preparations used for clinical transplants. EM allowed an accurate identification and quantification of cell types with measured cell number fractions (mean±s.e.m.) of 35.6±2.1% β-cells, 12.6±1.0% non-β-islet cells (48.3±2.6% total islet cells), 22.7±1.5% duct cells, and 25.3±1.8% acinar cells. Of the islet cells, 73.6±1.7% were β-cells. For comparison with the literature, estimates of cell number fraction, cell volume, and extracellular volume were combined to convert number fraction data to volume fractions applicable to cells, islets, and the entire preparation. The mathematical framework for this conversion was developed. By volume, β-cells were 86.5±1.1% of the total islet cell volume and 61.2±0.8% of intact islets (including the extracellular volume), which is similar to that of islets in the pancreas. Our estimates produced 1560±20 cells in an islet equivalent (volume of 150-μm diameter sphere), of which 1140±15 were β-cells. To test whether LM analysis of the same tissue samples could provide reasonable estimates of purity of the islet preparations, volume fraction of the islet tissue was measured on thin sections available from 27 of the clinical preparations by point counting morphometrics. Islet purity (islet volume fraction) of individual preparations determined by LM and EM analyses correlated linearly with excellent agreement (R²=0.95). However, islet purity by conventional dithizone staining was substantially higher with a 20-30% overestimation. Thus, both EM and LM provide accurate methods to determine the cell composition of human islet preparations and can help us understand many of the discrepancies of islet composition in the literature.

Figure 1

Identification of islet and non-islet tissue by light microscopy with plastic sections. Freshly isolated islet tissue is characterized by its cordlike pattern around vascular spaces (white areas) (A-C). These spaces partially collapse within 24 hr of culture at 37°C (D-G). Initially the vascular spaces of fresh human islets comprise about 14% of the islet volume (Pisania et al, submitted). Acinar cells (C, F,G) are distinguishable from the islets by their large zymogen granules (dark blue); the small terminal ducts (homogenous light blue) are seen surrounded by the acinar cells in these exocrine (ex) clumps (C,F,H). The exocrine clumps are initially compact (C) and do not show volume change with 24 hrs culture (G). Panel F shows necrosis of islet even after 24 hrs in culture. Toluidine blue stained one μm plastic sections of purified human islet preparations. Magnification bar = 50 μm.

Figure 2

Electron micrographs of pellets of purified islet preparations showing characteristics of the different cell types. (A) β-cells can be definitively identified by electron dense granules, often with crystals, with space between the granule limiting membrane and the hormone giving a typical “halo.” (B) Non-β-cells have granules without halos: the glucagon producing α-cells have homogenous electron dense granules; the somatostatin producing δ-cells are less homogeneous in density of the granules. (C) For the exocrine tissue, the acinar cells contain large dense zymogen granules and large amount of stacked ER whereas the ductal cells contain few organelles, inclusions or granules.

Figure 3

Frequency distribution of the vascular void volume fraction Φ_VI by LM for 27 freshly isolated clinical preparations.

Figure 4

Volume fraction (purity) data for individual islet preparations estimated by visual impressions of DTZ-stained preparations are plotted versus volume fraction estimated from cell composition as determined by EM together with estimates of volume per cell and extracellular volume fractions using Equation (3). The solid line is the line of identity. Data are from all 33 clinical islet preparations.

Figure 5

Calculated islet volume fraction by EM is plotted against the measured islet volume fractions by LM for 27 freshly isolated clinical preparations. The dashed line is the line of identity. The calculated islet volume fraction by EM correlates linearly with that measured by LM. Linear regression of the data gives a correlation coefficient R² = 0.95 for all data and R² = 0.97 without three data points for (Φ_I)_LM less than 0.3.

Figure 6

Estimated islet volume fraction by DTZ staining is plotted against the measured islet volume fractions by LM for 27 freshly isolated clinical preparations. The dashed line is the line of identity. In many cases, the measurement from DTZ staining was much higher than that from LM point counting and provided a gross overestimation of islet purity. Linear regression of the data gives a correlation coefficient R² = 0.67.

Figure 7

Frequency distribution of the islet volume fraction by (A) EM, (B) LM, and (C) DTZ staining for 27 freshly isolated clinical preparations.

Figure 8

Volume definitions and relationships in islet preparations. Other cells refer to the endothelial and connective tissue cells as well as cells that could not be classified.