Title: Embedded-microstructures arrays for single-cell sorting and collecting of lymphocytes following HIV latency reactivation
Abstract:
The study of single cells has recently accelerated in many fields, oncology, neuroscience, virology, and immunology, among others.Bulk studies provided excellent knowledge, but much information was lost when collecting data from cell populations. Different cell-separation platforms have been developed to comprehend single-cell heterogeneity. Microwell-based devices are among the most outstanding technologies for single-cell analysis due to their simplicity. We describe a new approach to manufacture microarrays. Depending on the deposition parameters, materials, and topography, embedded- microstructures arrays (EMA) were fabricated. These arrays possess optically transparent microwells with collectible magnetic microcarriers upon which the cells are cultured and screened using standard imaging techniques. EMA enables time-resolved imaging of cells combined with efficient isolation of target cells. Cultured cells can be identified by many selection criteria, including fluorescence intensity that changes over time. An individual carrier containing a target cell can be efficiently released and collected for genomic analysis using magnetic collections. In the current work, time-resolved assays will be carried out in order to evaluate the dynamics of HIV latency.
HIV-infected CD4+ T cells comprise a latent viral reservoir that can be reactivated using latency reversal agents (LRAs). Since HIV reactivation is not uniform across an entire population of latent cells, approaches to track reactivation within a single cell are critical to develop new strategies to eradicate HIV reservoirs. Our microarray platform was used to understand gene expression in newly reactivated HIV-infected cells compared to uninfected cells. The arrays used in this work are composed of 19,600 microcarriers with dimensions of 100 x 100 μm (W x L), 60 μm depth, and 50 μm spacing between the microrafts. We tracked mCherry and GFP expression of latently infected cells following exposure to three latency reversing agents (LRAs), prostratin, vorinostat, and iBET151. After single-cell analysis, reactivated latent cells were released to study transcriptional heterogeneity by performing single-cell RNA-sequencing. These results will advance our conceptual understanding of HIV reactivation dynamics.
