Nondestructive, quantitative viability analysis of 3D tissue cultures using machine learning image segmentation

Kylie J. Trettner, Jeremy Hsieh, Weikun Xiao*, Jerry S.H. Lee, Andrea M. Armani*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review


Ascertaining the collective viability of cells in different cell culture conditions has typically relied on averaging colorimetric indicators and is often reported out in simple binary readouts. Recent research has combined viability assessment techniques with image-based deep-learning models to automate the characterization of cellular properties. However, further development of viability measurements to assess the continuity of possible cellular states and responses to perturbation across cell culture conditions is needed. In this work, we demonstrate an image processing algorithm for quantifying features associated with cellular viability in 3D cultures without the need for assay-based indicators. We show that our algorithm performs similarly to a pair of human experts in whole-well images over a range of days and culture matrix compositions. To demonstrate potential utility, we perform a longitudinal study investigating the impact of a known therapeutic on pancreatic cancer spheroids. Using images taken with a high content imaging system, the algorithm successfully tracks viability at the individual spheroid and whole-well level. The method we propose reduces analysis time by 97% in comparison with the experts. Because the method is independent of the microscope or imaging system used, this approach lays the foundation for accelerating progress in and for improving the robustness and reproducibility of 3D culture analysis across biological and clinical research.

Original languageEnglish
Article number016121
JournalAPL Bioengineering
Issue number1
StatePublished - 1 Mar 2024
Externally publishedYes


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