Single-cell microscopy of suspension cultures using a microfluidics-assisted cell screening platform

Studies that rely on fluorescence imaging of nonadherent cells that are cultured in suspension, such as E. coli, are often hampered by trade-offs that must be made between data throughput and imaging resolution. A platform for microfluidics-assisted cell screening (MACS) was develop by key scientists from Department of Systems Biology Harvard Medical School (Boston, Massachusetts, USA) that overcomes this trade-off by temporarily immobilizing suspension cells within a microfluidics chip. This enables high-throughput and automated single-cell microscopy for a wide range of cell types and sizes.

Studies that rely on fluorescence imaging of nonadherent cells that are cultured in suspension, such as E. coli, are often hampered by trade-offs that must be made between data throughput and imaging resolution. Flow cytometry has been very powerful in cell biology, allowing researchers to conveniently analyze millions of individual cells growing in standard batch cultures. However, the throughput comes at the cost of low sensitivity, meaning that many low-abundance fluorescent proteins cannot be reliably detected. Flow cytometry also provides only limited information (Tracy BP et al., 2012; Bongaerts RJ et al., 2002) both about spatial localization of molecules and organelles inside cells, and the sizes and shapes of the cells themselves. By contrast, microscopy is capable of detecting cellular components across a wider range of abundances while providing accurate estimates of cell size, morphology and spatial patterns (Cisse II et al., 2013). However, throughput is often much lower, making it difficult to accurately determine statistical properties, detect rare cell states or condition the properties of interest to other measured properties (e.g., the distribution of protein numbers given a certain cell size). Microscopy also has other limitations. Cells often remain under the microscope for such long time periods—especially when gathering statistics—that their properties can change, making it difficult to compare the results to the batch culture. This problem has been partly addressed by using microfluidic growth chambers (Okumus B et al., 2014), which have been tremendously successful in the past few years, increasing throughput substantially and sometimes achieving a uniformity of growth conditions that is comparable to batch culture (Wang P et al., 2010). Okumus B and colleagues from Department of Systems Biology of Harvard Medical School (Boston, Massachusetts, USA) developed a platform for microfluidics-assisted cell screening (MACS) that overcomes this trade- off by temporarily immobilizing suspension cells within a microfluidics chip. MACS works by injecting cells through a flow channel, and gently compressing the ceiling of the channel. This enables high-throughput and automated single-cell microscopy for a wide range of noonadherent cell types and sizes. As cells can be rapidly sampled directly from a suspension culture, MACS bypasses the need for sample preparation, and therefore allows measurements without perturbing the native cell physiology. The setup can also be integrated with complex growth chambers, and can be used to enrich or sort the imaged cells. Furthermore, MACS facilitates the visualization of individual cytoplasmic fluorescent proteins (FPs) in E. coli, allowing low-abundance proteins to be counted using standard total internal reflection fluorescence (TIRF) microscopy. Finally, MACS can be used to impart mechanical pressure for assessing the structural integrity of individual cells and their response to mechanical perturbations, or to make cells take up chemicals that otherwise would not pass through the membrane. For the same purpose, CellDynamics has developed a cutting-edge technology, named CellViewer, for real-time monitoring of floating cells in a liquid medium. With the CellViewer, scientists can obtain detailed information on dynamic biological processes at the cellular and multicellular levels. By combining fluorescence time-lapse imaging, automated focal adjustments, environmental culture control and microfluidic media exchange technologies, CellViewer provides proper experimental conditions for continuous observations of single cell suspensions and cellular aggregates. These properties are optimal for applications ranging from basic research, pharmaceutical in vitro testing and in vitro fertilization.

Figure 6. Capturing rare phenotypes and their retrieval using MACS. (a) Cells continuously flowing through the field of view in the half-open state. Scale bar, 25 μm. (b) The cell of interest—an RFP-expressing cell, which we diluted by a factor of 100,000 using GFP-expressing cells—is captured in the closed state and is detected (circled). Scale bar, 10 μm. (c) Minor modification of MACS enables cell retrieval. Masks for flow (top) and control (bottom) layers. Scale bars, 1 mm. The flow-focusing feature is shown within the semitransparent gray box. This feature is optional, and allows the cells to trickle through the central region of the flow channel and prevent cell accumulation at the channel sides. It also allows adjustment of the cell density on the FOV: when the side streams (of media) are stronger, the middle stream becomes thinner, thereby diluting the cells at the FOV. (d) Schematic of cell retrieval. The volume that is trapped when valves 1–4 are closed (with the control valve open) is shown by the red dashed line. (e) The captured cell of interest in Figure 6b (circled) within the trapped volume, which is outlined by the red dashed line. Overlaid bright-field and RFP fluorescence images are shown. Scale bar, 40 μm. (f) When we collected the trapped volume using the oil phase, grew cells overnight and imaged them on the agar pad, the RFP-expressing cells were enriched. This scheme allows for the immediate retrieval of cells at low cell densities. At high densities, a second round is necessary to achieve 100% purity. Scale bar, 2 μm. Image adapted with permission from ref. 12, Nature Publishing Group from Okumus B et al., 2018. Single-cell microscopy of suspension cultures using a microfluidics-assisted cell screening platform. Nat Protoc. 2018 Jan;13(1):170-194).

References

  • Tracy, B.P., Gaida, S.M. & Papoutsakis, E.T. Flow cytometry for bacteria: enabling metabolic engineering, synthetic biology and the elucidation of complex phenotypes. Curr. Opin. Biotechnol. 21, 85–99 (2010).
  • Bongaerts, R.J., Hautefort, I., Sidebotham, J.M. & Hinton, J.C. Green fluorescent protein as a marker for conditional gene expression in bacterial cells. Methods Enzymol. 358, 43–66 (2002).
  • Cisse, I.I. et al. Real-time dynamics of RNA polymerase II clustering in live human cells. Science 341, 664–667 (2013).
  • Okumus, B., Yildiz, S. & Toprak, E. Fluidic and microfluidic tools for quantitative systems biology. Curr. Opin. Biotechnol. 25, 30–38 (2014).
  • Wang, P. et al. Robust growth of Escherichia coli. Curr. Biol. 20, 1099–1103 (2010).
  • Okumus B et al., 2018. Single-cell microscopy of suspension cultures using a microfluidics-assisted cell screening platform. Nat Protoc. 2018 Jan;13(1):170-194).

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