RESUMO
Despite intense scrutiny of the molecular interactions between natural killer (NK) and target cells, few studies have been devoted to dissection of the basic functional heterogeneity in individual NK cell behavior. Using a microchip-based, time-lapse imaging approach allowing the entire contact history of each NK cell to be recorded, in the present study, we were able to quantify how the cytotoxic response varied between individual NK cells. Strikingly, approximately half of the NK cells did not kill any target cells at all, whereas a minority of NK cells was responsible for a majority of the target cell deaths. These dynamic cytotoxicity data allowed categorization of NK cells into 5 distinct classes. A small but particularly active subclass of NK cells killed several target cells in a consecutive fashion. These "serial killers" delivered their lytic hits faster and induced faster target cell death than other NK cells. Fast, necrotic target cell death was correlated with the amount of perforin released by the NK cells. Our data are consistent with a model in which a small fraction of NK cells drives tumor elimination and inflammation.
Assuntos
Movimento Celular/imunologia , Células Matadoras Naturais/classificação , Células Matadoras Naturais/citologia , Ativação Linfocitária/imunologia , Linfócitos T Citotóxicos/classificação , Linfócitos T Citotóxicos/citologia , Apoptose/imunologia , Comunicação Celular/imunologia , Degranulação Celular/imunologia , Células HEK293 , Humanos , Imunofenotipagem , Células Matadoras Naturais/imunologia , Procedimentos Analíticos em Microchip , Modelos Biológicos , Necrose/imunologia , Linfócitos T Citotóxicos/imunologia , Imagem com Lapso de TempoRESUMO
We present a novel microwell array platform suited for various cell-imaging assays where single cell resolution is important. The platform consists of an exchangeable silicon-glass microchip for cell biological applications and a custom made holder that fits in conventional microscopes. The microchips presented here contain arrays of miniature wells, where the well sizes and layout have been designed for different applications, including single cell imaging, studies of cell-cell interactions or ultrasonic manipulation of cells. The device has been designed to be easy to use, to allow long-term assays (spanning several days) with read-outs based on high-resolution imaging or high-content screening. This study is focused on screening applications and an automatic cell counting protocol is described and evaluated. Finally, we have tested the device and automatic counting by studying the selective survival and clonal expansion of 721.221 B cells transfected to express HLA Cw6-GFP compared to untransfected 721.221 B cells when grown under antibiotic selection for 3 days. The device and automated analysis protocol make up the foundation for development of several novel cellular imaging assays.
Assuntos
Citometria de Fluxo/instrumentação , Análise em Microsséries/instrumentação , Análise em Microsséries/métodos , Linfócitos B/citologia , Contagem de Células , Técnicas de Cultura de Células/métodos , Linhagem Celular , Desenho de Equipamento , Citometria de Fluxo/métodos , Humanos , Microscopia Eletrônica , Microtecnologia , Análise de Célula Única/instrumentação , Análise de Célula Única/métodos , TransfecçãoRESUMO
We present a droplet-based microfluidic platform to automatically track and characterize the behavior of single cells over time. This high-throughput assay allows encapsulation of single cells in micro-droplets and traps intact droplets in arrays of miniature wells on a PDMS-glass chip. Automated time-lapse fluorescence imaging and image analysis of the incubated droplets on the chip allows the determination of the viability of individual cells over time. In order to automatically track the droplets containing cells, we developed a simple method based on circular Hough transform to identify droplets in images and quantify the number of live and dead cells in each droplet. Here, we studied the viability of several hundred single isolated HEK293T cells over time and demonstrated a high survival rate of the encapsulated cells for up to 11 hours. The presented platform has a wide range of potential applications for single cell analysis, e.g. monitoring heterogeneity of drug action over time and rapidly assessing the transient behavior of single cells under various conditions and treatments in vitro.
Assuntos
Técnicas Analíticas Microfluídicas/métodos , Análise de Célula Única/métodos , Automação , Sobrevivência Celular , Dimetilpolisiloxanos/química , Células HEK293 , Humanos , Técnicas Analíticas Microfluídicas/instrumentação , Microscopia Confocal , Água/químicaRESUMO
Natural killer (NK) cells kill virus-infected or cancer cells through the release of cytotoxic granules into a tight intercellular contact. NK cell populations comprise individual cells with varying sensitivity to distinct input signals, leading to disparate responses. To resolve this NK cell heterogeneity, we have designed a novel assay based on ultrasound-assisted cell-cell aggregation in a multiwell chip allowing high-resolution time-lapse imaging of one hundred NK-target cell interactions in parallel. Studying human NK cells' ability to kill MHC class I deficient tumor cells, we show that approximately two thirds of the NK cells display cytotoxicity, with some NK cells being particularly active, killing up to six target cells during the assay. We also report that simultaneous interaction with several susceptible target cells increases the cytotoxic responsiveness of NK cells, which could be coupled to a previously unknown regulatory mechanism with implications for NK-mediated tumor elimination.
Assuntos
Comunicação Celular , Separação Celular/instrumentação , Citometria de Fluxo/instrumentação , Células Matadoras Naturais/fisiologia , Neoplasias Experimentais/fisiopatologia , Sonicação/instrumentação , Análise Serial de Tecidos/instrumentação , Sobrevivência Celular , Células Cultivadas , Desenho de Equipamento , Análise de Falha de Equipamento , Humanos , Células Matadoras Naturais/citologia , Neoplasias Experimentais/patologiaRESUMO
We present a simple method for rapid and automatic characterization of lymphocyte migration from time-lapse fluorescence microscopy data. Time-lapse imaging of natural killer (NK) cells in vitro and in situ, both showed that individual cells transiently alter their migration behavior. Typically, NK cells showed periods of high motility, interrupted by transient periods of slow migration or almost complete arrests. Analysis of in vitro data showed that these periods frequently coincided with contacts with target cells, sometimes leading to target cell lysis. However, NK cells were also commonly observed to stop independently of contact with other cells. In order to objectively characterize the migration of NK cells, we implemented a simple method to discriminate when NK cells stop or have low motilities, have periods of directed migration or undergo random movement. This was achieved using a sliding window approach and evaluating the mean squared displacement (MSD) to assess the migration coefficient and MSD curvature along trajectories from individual NK cells over time. The method presented here can be used to quickly and quantitatively assess the dynamics of individual cells as well as heterogeneity within ensembles. Furthermore, it may also be used as a tool to automatically detect transient stops due to the formation of immune synapses, cell division or cell death. We show that this could be particularly useful for analysis of in situ time-lapse fluorescence imaging data where most cells, as well as the extracellular matrix, are usually unlabelled and thus invisible.