Design and analysis of a long-term live-cell imaging chamber for tracking cellular dynamics within cultured human islets of Langerhans.
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.
Moogk
Duane
D
Department of Chemical Engineering, University of Waterloo, Waterloo, Ontario, Canada.
Hanley
Stephen
S
Ramunas
John
J
Blaylock
April
A
Skorepova
Jana
J
Rosenberg
Lawrence
L
Jervis
Eric
E
D018929:Cell Culture Techniques / Q000295:instrumentation* / Q000379:methods; D002478:Cells, Cultured; D017076:Computer-Aided Design; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005434:Flow Cytometry / Q000295:instrumentation* / Q000379:methods; D005947:Glucose / Q000378:metabolism*; D006801:Humans; D007090:Image Interpretation, Computer-Assisted / Q000295:instrumentation* / Q000379:methods; D007515:Islets of Langerhans / Q000166:cytology* / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*
D005947:Glucose; D010100:Oxygen