Published April 2024 | Published
Journal Article Open

A novel approach to determine the critical survival threshold of cellular oxygen within spheroids via integrating live/dead cell imaging with oxygen modeling

  • 1. ROR icon California Institute of Technology
  • 2. ROR icon City Of Hope National Medical Center

Abstract

Defining the oxygen level that induces cell death within 3-D tissues is vital for understanding tissue hypoxia; however, obtaining accurate measurements has been technically challenging. In this study, we introduce a noninvasive, high-throughput methodology to quantify critical survival partial oxygen pressure (pO2) with high spatial resolution within spheroids by using a combination of controlled hypoxic conditions, semiautomated live/dead cell imaging, and computational oxygen modeling. The oxygen-permeable, micropyramid patterned culture plates created a precisely controlled oxygen condition around the individual spheroid. Live/dead cell imaging provided the geometric information of the live/dead boundary within spheroids. Finally, computational oxygen modeling calculated the pO2 at the live/dead boundary within spheroids. As proof of concept, we determined the critical survival pO2 in two types of spheroids: isolated primary pancreatic islets and tumor-derived pseudoislets (2.43 ± 0.08 vs. 0.84 ± 0.04 mmHg), indicating higher hypoxia tolerance in pseudoislets due to their tumorigenic origin. We also applied this method for evaluating graft survival in cell transplantations for diabetes therapy, where hypoxia is a critical barrier to successful transplantation outcomes; thus, designing oxygenation strategies is required. Based on the elucidated critical survival pO2, 100% viability could be maintained in a typically sized primary islet under the tissue pO2 above 14.5 mmHg. This work presents a valuable tool that is potentially instrumental for fundamental hypoxia research. It offers insights into physiological responses to hypoxia among different cell types and may refine translational research in cell therapies.

Copyright and License

© 2024 the American Physiological Society.

Acknowledgement

The authors thank Drs. Colin Cook and Nicholas Scianmarello for the insightful discussion. The authors also thank Dr. Sung Hee Kil for critical reading and editing of the manuscript.

Contributions

K.-M.S., H. Kato., and H. Komatsu conceived and designed research; K.-M.S., H. Kato, N.G, and H. Komatsu performed experiments; K.-M.S., H. Kato, and H. Komatsu analyzed data; K.-M.S., H. Kato, and H. Komatsu interpreted results of experiments; K.-M.S., H. Kato, and H. Komatsu prepared figures; K.-M.S. and H. Kato drafted manuscript; K.-M.S., H. Kato., Y.-C.T., and H. Komatsu edited and revised manuscript; K.-M.S., H. Kato, F.K., Y.-C.T., and H. Komatsu approved final version of manuscript.

Funding

This work was supported by Nora Eccles Treadwell Foundation, No Grant Number (to H. Komatsu); National Institutes of Health, Grant Number: R03DK129958-01 (to H. Komatsu); and Juvenile Diabetes Research Foundation, Grant Number: 3-SRA-2021-1073-S-B (to H. Komatsu).

Supplemental Material

Supplemental Figs. S1–S3: https://doi.org/10.6084/m9.figshare.24986859.

Files

shang-et-al-2024-a-novel-approach-to-determine-the-critical-survival-threshold-of-cellular-oxygen-within-spheroids-via.pdf

Additional details

Created:
December 4, 2024
Modified:
December 4, 2024