Design and analysis of a long‐term live‐cell imaging chamber for tracking cellular dynamics within cultured human islets of Langerhans

Abstract

A means of expanding islet cell mass is urgently needed to supplement the limited availability of donor islets of Langerhans for transplant. Live cell imaging of human islets in culture has the potential to identify the specific cells and processes involved in islet expansion. A novel imaging chamber was developed to facilitate long-term three-dimensional imaging of human islets during transformation. Islets have been induced to transform into duct-like epithelial cystic structures and revert back to glucose responsive endocrine cells under appropriate conditions (Jamal et al. Cell Death Differ. 2005 12:702-712). Here we aim to further our understanding by characterizing the process at a single cell level over time-essentially constructing a high resolution recorded history of each cell and its progeny during transformation and reversion. The imaging chamber enables high resolution imaging of three-dimensional islets while maintaining the structure of the islet cells and intercellular matrix components. A mathematical model was developed to validate the imaging chamber design by determining the required chamber dimensions to avoid introduction of oxygen and nutrient transport limitations. Human islets were embedded in collagen in the imaging chamber and differential interference contrast time course images were obtained at 3 min intervals. Immunofluorescent imaging confirmed that islet phenotype was maintained for at least 5 days during imaging. Analysis of the time courses confirms our ability to identify and track individual cells over time and to observe cell death and phenotype transformation in isolated human islets.

Human ESC colony formation is dependent on interplay between self‐renewing hESCs and unique precursors responsible for niche generation

“…Collected hESC aggregates were gently triturated to clumps of approximately 10–20 cells and seeded into Matrigel‐coated gap‐chambers. Gap chambers were assembled and loaded as previously described (15). Gap chambers were precoated with 1:30 Matrigel in KO‐DMEM containing 4.7 μm polystyrene beads (Polysciences) at 10 6 beads/ml for 2–5 h uncovered at room temperature, rinsed gently and then coated either at room temperature for 1 h or overnight at 4°C with 1:15 Matrigel.…”

“…Here we have used live cell imaging in combination with a previously reported culture system (12–15), cell tracking and phenotype scoring to investigate hESC niche regulation. Our results show a previously unappreciated level of morphological and behavioral heterogeneity at the hESC colony perimeter that corresponds to presence or absence of adjacent co‐transferred hdFs.…”

In vivo imaging of murine endocrine islets of Langerhans with extended-focus optical coherence microscopy

“…This principle could be adapted to reveal the pancreatic blood flow and provide additional functional variables. The sensitivity of xfOCM should prove useful to monitor changes in beta cell number-for example, during islet regeneration [30] or upon islet transplantation to visualise the islets and their vascularisation in the recipient. Analysing dispersion and spectral absorption characteristics in the sample could add further functional contrast such as variations in metabolite concentrations [31,32].…”

Constrained watershed method to infer morphology of mammalian cells in microscopic images

“…Cell imaging chambers were custom‐made and loaded, as previously described in Ref. (20) and summarized as follows. A small strip (10 × 2 mm) of glass Fisher microscope cover glass (22 × 50 mm) was washed with 10% NaOH and 1% HCl, dipped in methanol, and dried under nitrogen gas.…”