{"database":"biostudies-literature","file_versions":[],"scores":null,"additional":{"submitter":["Razooky BS"],"funding":["NIAID NIH HHS","NHLBI NIH HHS","NIGMS NIH HHS","NIH HHS"],"pagination":["4305-12"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/S-EPMC3589574"],"repository":["biostudies-literature"],"omics_type":["Unknown"],"volume":["12(21)"],"pubmed_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."],"journal":["Lab on a chip"],"pubmed_title":["Microwell devices with finger-like channels for long-term imaging of HIV-1 expression kinetics in primary human lymphocytes."],"pmcid":["PMC3589574"],"funding_grant_id":["GM083395","GM084332","P30 AI027763","T32 GM008326","AI090935","P01 HL078784","DP2OD00667","U19 AI096113","AI096113","DP2 OD006677","R01 GM084332","GM08326","P01 AI090935","K25 GM083395","HL078784"],"pubmed_authors":["Weinberger LS","Razooky BS","Terry VH","Gutierrez E","Spina CA","Groisman A"],"additional_accession":[]},"is_claimable":false,"name":"Microwell devices with finger-like channels for long-term imaging of HIV-1 expression kinetics in primary human lymphocytes.","description":"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.","dates":{"release":"2012-01-01T00:00:00Z","publication":"2012 Nov","modification":"2020-10-29T13:02:05Z","creation":"2019-03-27T01:05:41Z"},"accession":"S-EPMC3589574","cross_references":{"pubmed":["22976503"],"doi":["10.1039/c2lc40170c"]}}