title: Microwell devices with finger-like channels for long-term imaging of HIV-1 expression kinetics in primary human lymphocytes.
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.
authors.affiliation: Department of Chemistry and Biochemistry, University of California, San Diego, CA 92023, USA.
authors.forename: Brandon S
authors.initials: BS
authors.lastname: Razooky
authors.forename: Edgar
authors.initials: E
authors.lastname: Gutierrez
authors.forename: Valeri H
authors.initials: VH
authors.lastname: Terry
authors.forename: Celsa A
authors.initials: CA
authors.lastname: Spina
authors.forename: Alex
authors.initials: A
authors.lastname: Groisman
authors.forename: Leor S
authors.initials: LS
authors.lastname: Weinberger
mesh_terms: D015496:CD4-Positive T-Lymphocytes / Q000166:cytology / Q000821:virology*; D002478:Cells, Cultured; D015967:Gene Expression Regulation, Viral; D049452:Green Fluorescent Proteins / Q000096:biosynthesis*; D015497:HIV-1 / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D007700:Kinetics; D059010:Single-Cell Analysis / Q000295:instrumentation; D059008:Time-Lapse Imaging / Q000295:instrumentation*
chemical_list: D049452:Green Fluorescent Proteins