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

Pisania A, Weir GC, O'Neil JJ, Omer A, Tchipashvili V, Lei J, Colton CK, Bonner-Weir S - Lab. Invest. (2010)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA. 02139-4307

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.

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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.
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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.

Mentions: A 0.5 ml aliquot from the final islet preparation was fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, osmicated, divided into two samples, and embedded in plastic (Araldite) in the Joslin Advanced Microscopy Core. Thin (1 μm) sections were stained with toluidine blue for initial evaluation of purity and quality of islets. Secretory granules of islet endocrine cells were too small to be seen with LM, but the zymogen granules of acinar cells were very evident (Figure 1), which allowed acinar contamination to be easily identified. Ultrathin sections to be taken to EM were cut from the same blocks. Sixteen micrographs per sample were taken systematically to cover the section, using 1900× magnification to give a total of 32 micrographs per islet preparation. A magnification of 1900× provided adequate sampling with a minimum of 500 cells; with photographic printing (final magnification 4375×), the granule morphology of the cells could be distinguished on the micrographs. Cell boundaries on each micrograph were determined to indicate the number of cells; then each cell was assigned to a category of β, non-β endocrine, acinar, or ductal cells. Acinar cells, islet cells (β, and the non- β cells α, δ and PP), and duct cells could be definitively identified, and thus, cell composition determined (Figure 2). Occasional dead cells or endothelial cells were also identified and characterized as “other”. The resulting cell composition was based on number, not volume, of cells counted from both samples and yielded the number fraction of each category. Islet volume fraction measurements by LM were made retrospectively on thin sections that were available for 27 of the 33 freshly isolated clinical preparations.


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

Pisania A, Weir GC, O'Neil JJ, Omer A, Tchipashvili V, Lei J, Colton CK, Bonner-Weir S - Lab. Invest. (2010)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2966538&req=5

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.
Mentions: A 0.5 ml aliquot from the final islet preparation was fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, osmicated, divided into two samples, and embedded in plastic (Araldite) in the Joslin Advanced Microscopy Core. Thin (1 μm) sections were stained with toluidine blue for initial evaluation of purity and quality of islets. Secretory granules of islet endocrine cells were too small to be seen with LM, but the zymogen granules of acinar cells were very evident (Figure 1), which allowed acinar contamination to be easily identified. Ultrathin sections to be taken to EM were cut from the same blocks. Sixteen micrographs per sample were taken systematically to cover the section, using 1900× magnification to give a total of 32 micrographs per islet preparation. A magnification of 1900× provided adequate sampling with a minimum of 500 cells; with photographic printing (final magnification 4375×), the granule morphology of the cells could be distinguished on the micrographs. Cell boundaries on each micrograph were determined to indicate the number of cells; then each cell was assigned to a category of β, non-β endocrine, acinar, or ductal cells. Acinar cells, islet cells (β, and the non- β cells α, δ and PP), and duct cells could be definitively identified, and thus, cell composition determined (Figure 2). Occasional dead cells or endothelial cells were also identified and characterized as “other”. The resulting cell composition was based on number, not volume, of cells counted from both samples and yielded the number fraction of each category. Islet volume fraction measurements by LM were made retrospectively on thin sections that were available for 27 of the 33 freshly isolated clinical preparations.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA. 02139-4307

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.

Show MeSH
Related in: MedlinePlus