T18 - Data Processing Pipeline to Analyze Calcium Signaling Dynamics in Lymphatic Endothelial Cells

Introduction: : Intracellular calcium signaling plays a crucial role in many key cellular processes, such as proliferation, metabolism, migration, etc.1-4 In response to external signals, cells are capable of drastically increasing the flux of calcium ions from either extracellular space or intracellular calcium storage in organelles such as the endoplasmic reticulum.5-6 Calcium signaling is a ubiquitous aspect of many intracellular communication pathways because of a large set of signals, receptors, and transporters that can be applied to multiple cellular processes.7,8 Understanding calcium signaling is especially important in tissue engineering and other bioengineering applications that focus on population-wide cell communication. Calcium also serves as an intercellular communication tool in many cells, aiding in self-organizational behavior of cells often seen in organogenesis.9-11a Therefore, there is a compelling need to advance our ability to understand and manipulate large-scale calcium signaling dynamics for bioengineering applications.
We have established a data processing pipeline that is designed around analyzing intracellular calcium oscillation patterns in non-excitable cells, such as endothelial cells which have distinct internal calcium oscillation pattern depending on the state of the cell. By creating a pipeline that determines if a cell is defined as spiking or non-spiking at specific time points, that can then be analyzed for the frequency of signaling and cross-correlation values. This data processing pipeline will be used to continue to develop a deep-learning solution to characterize the intracellular calcium oscillation behavior in endothelial cells based on the morphology and cell-communication networks, which will have broad implications for various approaches in tissue engineering.

Materials and

Methods: : For this pipeline (Fig 1) we take raw fluorescence image stack in tiff format taken at between 4x and 10 magnifications as input. The input images are fluorescent images of cells of mesodermal lineage that express fluorescent signal intensity shift that correspond to cytoplasmic calcium. After undergoing preprocessing, the cells are segmented so that data can be extracted based of fluorescent intensity. After, we can detect for frequency, amplitude, and correlation depending on what the study indicates a need for. In this study, we utilized Calbryte 520AM dye, a robust calcium indicator dye that is non-fluorescent unless bound with a calcium ion inside the cell. The cells used for imaging were passage 7 Blood Endothelial Cells and Lymphatic Endothelial Cells as well as differentiated endothelial cells via a modified protocol of the dEC protocol by Harding and colleagues.

Results, Conclusions, and Discussions:: Utilizing this proposed pipeline, we are able to characterize intracellular calcium signaling characteristics in relation to population signaling dynamics in various conditions. The pipeline can detect shifts in frequency, and it was shown through the addition of VEGF to the endothelial cell populations (Fig 2). It is also possible to find correlation between the spiking of the cells which may indicate intercellular communication, and we can use this to compare the correlation peaks and shifts to indicate what environmental stimulators can increase the cellular communication (Fig 3).

Acknowledgements and/or References (Optional):: References
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