Comprehensive Analysis of Human Pancreatic Islets Using Flow and Laser Scanning Cytometry

Assessing islet cellular composition and β cell viability using Flow Cytometry (FC) and Laser Scanning Cytometry (LSC) may aid in determining the transplant quality of islets.Human islets (2500 IEQ, n = 44, purity ≥80%) dissociated into a single cell suspension were stained with ductal marker CA19, with Newport Green (NG) and FluoZin3 (FL3) for β-cell identification, with TMRE to assess mitochondrial membrane potential, with DAPI to identify live vs. dead cells, and with Annexin-V/DAPI to differentiate apoptotic and necrotic cells. For LSC, cell preparations (n = 9) were stained for insulin (β-cells), glucagon (α-cells), somatostatin (δ cells), and pancreatic polypeptide (ppp cells). Fluorescence microscopy (EtBr/FDA) and insulin response were also measured. DAPI− staining was 73.78% ± 1.37, while EtBr/FDA was 96% ± 0.48. 52.5% ± 3.73 of all cells were NG+, of which 58.08% ± 2.61 were NG+/TMRE+. Annexin-V/DAPI staining (n = 26) showed 13.8% ± 0.89 apoptotic, 27.2% ± 2.0 necrotic, and 51.9% ± 2.22 live cells. 26.0% ± 5.19 of cells were CA19 positive (n = 17), of which 45.5% ± 4.37 were also TMRE+, and 5.2% ± 1.2 of the TMRE+ were also NG+/CA19+. NG and FL3 showed similar staining (n = 8). Comparison of short-term (≤2 days) versus long-term (≥3 days) culture showed similar TMRE+/NG+ averages, albeit lower percentages of live (36.4% vs 51.9%), and higher percentages of apoptotic (19.2% vs 13.8%) and necrotic cells (37.4% vs 27.2%) for long-term, as determined by Annexin-V staining. LSC resulted in 54.17% ± 4.62 β-cells, 33.33% ± 4.16 α-cells, 8.75% ± 2.5 δ-cells, and 3.75% ± 0.79 ppp cells. There is no significant difference between insulin positive cells and NG positive cells (P ≤ .55). FC and LSC provide valuable information about islet quality, which could potentially be used for evaluating islets prior to transplantation.