Arrays of bottomless picowells for the analysis of biological age using high-resolution imaging

About This Grant

Abstract Identification and validation of biomarkers of aging, which predict the lifespan and risk of disease better than chronological age and can help identify factors that accelerate aging and interventions that promote resilience, is the major goal of the Predictive Biomarkers Initiative of the NIA. DNA methylation (DNAm) clocks, which provide assessments of chronological and biological age, have attracted a lot of attention and have been incorporated into multiple commercial products. Nevertheless, the DNAm clocks are usually based on averages over large pools of cells of different types and extract age-related trends from noisy data. Recently, the Co-PI lab pioneered the analysis of cellular states, identities, and perturbations based on high-resolution confocal imaging of epigenetic marks in nuclei of individual immuno-labeled cells and applied this approach to aging. The imaging-based chromatin and epigenetic age (ImAge) analysis captures the principal changes in the spatial organization of chromatin and epigenetic marks that correlate with biological age. The cell-to-cell variability of the ImAge readouts increases with age, making it a separate metric of the process of aging and highlighting the value of obtaining the quantitative epigenetic signatures from single cells. ImAge measurements require only small amounts of Peripheral Blood Mononuclear Cells (PBMCs) that can potentially be obtained from fingerstick blood samples. To effectively implement ImAge for assessing the human biological age, PBMCs obtained from a fingerstick must be immuno-labeled and presented for high- resolution imaging with minimal cell loss. To meet this challenge, we propose a platform for single-cell capture, immuno-labeling, and high-resolution imaging of PBMCs in arrays of bottomless microscopic conical wells with picoliter volumes. When captured in a conical picowell, a round cell rests on the tilted sidewalls and remains motionless under moderate mechanical perturbations, including medium exchanges during the immuno- labeling. The major advantage of the proposed bottomless conical picowells is that the cell loading is by vertical flow through the picowells, thus minimizing the cell loss, increasing the proportion of picowells with single cells, and filtering out cell debris. We will develop and validate an ImAge platform based on arrays of bottomless picowells and fluidic perfusion devices. We will apply it to obtain the first ImAge dataset of human PBMCs from healthy donors of different ages. When fully developed and tested, the proposed bottomless picowell-based platform will enable cost-effective longitudinal tracking of biological age of individual patients to facilitate personalized medicine and to achieve a better understanding of how aging is affected by different life events, environmental changes, and medical interventions.