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Issue 21, 2012
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Microwell devices with finger-like channels for long-term imaging of HIV-1 expression kinetics in primary human lymphocytes

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Abstract

A major obstacle in the treatment of human immunodeficiency virus type 1 (HIV-1) is a sub-population of latently infected CD4+ T lymphocytes. The cellular and viral mechanisms regulating HIV-1 latency are not completely understood, and a promising technique for probing the regulation of HIV-1 latency is single-cell time-lapse microscopy. Unfortunately, CD4+ T lymphocytes rapidly migrate on substrates and spontaneously detach, making them exceedingly difficult to track, hampering single-cell level studies. To overcome these problems, we built microdevices with a three-level architecture. The devices contain arrays of finger-like microchannels to “corral” T-lymphocyte migration, round wells that are accessible to pipetting, and microwells connecting the microchannels with the round wells. T lymphocytes that are loaded into a well first settle into the microwells and then to microchannels by gravity. Within the microchannels, T lymphocytes are in favorable culture conditions because they are in physical contact with each other, under no mechanical stress, and fed from a large reservoir of fresh medium. Most importantly, T lymphocytes in the microchannels are not exposed to any flow and their random migration is restricted to a nearly one-dimensional region, greatly facilitating long-term tracking of multiple cells in time-lapse microscopy. The devices have up to nine separate round wells, making it possible to test up to nine different cell lines or medium conditions in a single experiment. Activated primary CD4+ T lymphocytes, resting primary CD4+ T lymphocytes, and THP-1 monocytic leukemia cells loaded into the devices maintained viability over multiple days. The devices were used to track the fluorescence level of individual primary CD4+ T lymphocytes expressing green fluorescent protein (GFP) for up to 60 hours (h) and to quantify single-cell gene-expression kinetics of four different HIV-1 variants. The kinetics of GFP expression from the lentiviruses in the primary CD4+ T lymphocytes agree with previous measurements of these lentiviral vectors in the immortalized Jurkat T lymphocyte cell line. The proposed devices offer a simple, robust approach to long-term single-cell studies of environmentally sensitive primary lymphocytes.

Graphical abstract: Microwell devices with finger-like channels for long-term imaging of HIV-1 expression kinetics in primary human lymphocytes

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Publication details

The article was received on 17 Feb 2012, accepted on 21 Aug 2012 and first published on 21 Aug 2012


Article type: Paper
DOI: 10.1039/C2LC40170C
Citation: Lab Chip, 2012,12, 4305-4312
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    Microwell devices with finger-like channels for long-term imaging of HIV-1 expression kinetics in primary human lymphocytes

    B. S. Razooky, E. Gutierrez, V. H. Terry, C. A. Spina, A. Groisman and L. S. Weinberger, Lab Chip, 2012, 12, 4305
    DOI: 10.1039/C2LC40170C

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