Identifying HIF activity in three-dimensional cultures of islet-like clusters.


PURPOSE Hypoxia is a major cause for failure of encapsulated islet grafts. Three-dimensional (3D) re-aggregation and hypoxic preconditioning are used to help overcome this obstacle. However, it is still difficult to identify hypoxic cells in a 3D system. We evaluate the efficacy of a fluorescent system for detecting HIF-1 activity in live β-cells. Identification of HIF-1 activity and correlation with insulin secretion and viability will allow for more informed implant construction and better prediction of post-transplantational function.
 METHODS MIN6 cells were infected with the marker virus and rotationally cultured to form clusters. Clusters were encapsulated in PEG hydrogels and incubated in 20%, 2%, or 1% O2. Gels were imaged daily for hypoxia marker signaling and for morphological observation. Daily GSIS was quantified by insulin ELSIA and cell viability was assessed by LIVE/DEAD staining.
 RESULTS Clusters cultured in 2% and 1% O2 displayed high levels of HIF activity compared to 20% O2 
clusters. 20% O2 clusters maintained viability and achieved a smooth, islet-like morphology by 
Day 14. Clusters in 2% and 1% O2 failed to associate cohesively and showed reduced viability. As a whole, constructs cultured in 20% O2 exhibited 10-fold higher GSIS than constructs in 2% and 1% O2.
 CONCLUSIONS Our marker is an effective approach for identifying cellular hypoxia in 3D cultures. β-cell clusters in 2% and 1% O2 are similarly affected by reduced oxygen tension, with HIF-1 activity correlating to reduced GSIS and impaired cell/cluster morphology. Simultaneous aggregative culture and hypoxic conditioning may not be beneficial to β-cell transplantation.

DOI: 10.5301/IJAO.5000193

Cite this paper

@article{Skiles2013IdentifyingHA, title={Identifying HIF activity in three-dimensional cultures of islet-like clusters.}, author={Matthew L. Skiles and Nathan Wilder and Suchit Sahai and James O. Blanchette}, journal={The International journal of artificial organs}, year={2013}, volume={36 3}, pages={175-83} }