Department of Chemistry and Biochemistry, University of California, San Diego, CA 92023, USA.
Lab Chip. 2012 Nov 7;12(21):4305-12. doi: 10.1039/c2lc40170c.
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.
治疗人类免疫缺陷病毒 1 型（HIV-1）的一个主要障碍是潜伏感染的 CD4(+)T 淋巴细胞亚群。调节 HIV-1 潜伏的细胞和病毒机制尚未完全阐明，探测 HIV-1 潜伏调节的一种有前途的技术是单细胞延时显微镜。不幸的是，CD4(+)T 淋巴细胞在基质上迅速迁移并自发脱落，使得它们极难追踪，阻碍了单细胞水平的研究。为了克服这些问题，我们构建了具有三级结构的微器件。这些设备包含阵列状的指状微通道，用于“圈养”T 淋巴细胞迁移，可用于移液的圆形井，以及连接微通道和圆形井的微井。装入井中的 T 淋巴细胞首先沉降到微井中，然后通过重力沉降到微通道中。在微通道中，T 淋巴细胞处于有利的培养条件下，因为它们彼此物理接触，不受机械应力，并且从大量新鲜培养基中获得营养。最重要的是，微通道中的 T 淋巴细胞不会暴露于任何流动中，它们的随机迁移被限制在几乎一维的区域，极大地促进了延时显微镜中多个细胞的长期跟踪。这些设备有多达九个独立的圆形井，可以在单个实验中测试多达九个不同的细胞系或培养基条件。装入设备的激活的原代 CD4(+)T 淋巴细胞、静止的原代 CD4(+)T 淋巴细胞和 THP-1 单核白血病细胞在多天内保持活力。该设备用于跟踪表达绿色荧光蛋白（GFP）的单个原代 CD4(+)T 淋巴细胞的荧光强度，长达 60 小时（h），并定量四种不同 HIV-1 变体的单细胞基因表达动力学。原代 CD4(+)T 淋巴细胞中慢病毒的 GFP 表达动力学与这些慢病毒载体在永生化 Jurkat T 淋巴细胞系中的先前测量结果一致。所提出的设备为长期单细胞环境敏感原代淋巴细胞研究提供了一种简单、稳健的方法。
