Volume reduction rate in human and rat pancreatic islets in culture after isolation

Volume reduction rate in human and rat pancreatic islets in culture after isolation Sarah Suergiu*1, 2, David Habart1, Klara Zacharovová1, Zuzana Berková1, Barbora Radochová3, Jiří Janáček3, Martin Čapek4, Frantisek Saudek5 [1] Institute of Clinical and Experimental Medicine, Prague, Czech Republic; [2] Charles University, Faculty of Science, Prague, Czech Republic; [3] Institute of Physiology of the Czech Academy of Sciences, Laboratory of Biomathematics, Prague, Czech Republic; [4] Institute of Molecular Genetics of the Czech Academy of Sciences, Light Microscopy Core Facility, Prague, Czech Republic; [5] Institute of Clinical and Experimental Medicine, Diabetes Centre, Prague, Czech Republic Conclusions Supported by the project National Institute for Research of Metabolic and Cardiovascular Diseases (Program EXCELES, ID Project No. LX22NPO5104) – Funded by the European Union – Next Generation EU, and by the project from Czech Ministry of Health (NU22-0100141). References: 1.Pisania, A. et al. Quantitative analysis of cell composition and purity of human pancreatic islet preparations. Lab. Investig. J. Tech. Methods Pathol. 90, 1661–1675 (2010); 2. Girman, P. et al. Rat Experimental Transplantation Surgery: A Practical Guide. (Springer International Publishing, Cham, 20153. Niclauss N et al. Computer-assisted digital image analysis to quantify the mass and purity of isolated human islets before transplantation. Transplantation. 2008 Dec;86(11):1603-1609 Acknowledgments Fig.1 Rat islets (A) Histogram of islet sizes at baseline (t₀). (B) Volume change dynamics over time. (C) Violin plot of islet volume ratio at 17 hours relative to t₀ (t₁₇h / t₀). Median: 0.88; upper whisker: 0.99; lower whisker: 0.66. Methods Results and discussion Rat islets Human Islets Human islets Rat (Wistar) islets2 Islet isolation -3h -1h -0.5h -6h -2h -0.5h Segmentation script Islet size from area3 d=2√(S/π) Application of Sphiracle volume model t0t0 t17h t17h t0 t17h Original Auto segmentation Sphiracle model Pancreas digestion Gradient purification Handpicking and transportation Timelapse, on-stage incubator microscope (Leica DMI8, 37°C, 5% CO2, complete medium): •first 3h: 1 image every 20 min •1 image per hour until end of experiment Pisania et al. (2010) reported an average 14% volume reduction in islets during culture1: •Islets volumes compared only at two discrete time points; •Conducted on histological samples of pooled isolated islets; •Evaluation performed at the population level, without tracking individual islets; •The dynamics of the volume reduction remains unknown This study aims to investigate the volumetric changes over the first hours post isolation in standard cell culture. Specifically: •life microscopy; •volume estimation from 2D projections; •individual islet tracking ; •rat vs human islets. Background Volume reduction ratio after 17 hours Aims Volume Dynamics of Islets: Approximately 50% of the total volume change occurs within the first 3 hours, indicating a two-phase dynamic behavior. Species Comparison: Human islet isolation procedures are typically longer and involve extended exposure to cold storage solutions, which may contribute to initial swelling upon rewarming. Variability Factors: High variability was observed among samples, independent of islet size. The isolation process may affect islets differently, potentially causing both vascular collapse (as reported by Pisania et al.) and cellular loss, as clearly visible in our time-lapse imaging. Study Limitations: •Automated segmentation may introduce artefacts, even after manual correction. Out of the original total of 133 islets, 19 were excluded because of the poor segmentation quality; •Prolonged incubation beyond 17 hours can lead to medium evaporation, limiting the feasibility of extended observation periods. Fig.2 Human islets (A) Histogram of islet sizes at baseline (t₀). (B) Volume change dynamics over time. (C) Violin plot of islet volume ratio at 15 hours relative to t₀ (t15h / t₀). Median: 0.82; upper whisker: 0.99; lower whisker: 0.59. A N = 65 islets N = 49 islets [email protected] Individual islet life microscopy tracing Volume estimation A BC B C 0.88 0.82 i01 i11 i01 i04 (B) Black line: fitted curve. White dotted line: mean volume ratio. Red area: standard deviation range. Colored dotted lines: representative examples of individual islets tracked over time. We observed islet volume reduction that is: •of differing magnitude between rat and human islets (14% and 19%, respectively), probably accounting for differences in the isolation techniques; •independent of initial islet size; •following a biphasic pattern, with 50% of the reduction occurring within 90 minutes post-isolation. Volume reduction ratio after 15 hours Volume reduction dynamics Volume reduction dynamics