GEN Exclusives

More »

Feature Articles

More »
Jul 1, 2012 (Vol. 32, No. 13)

Multiple Routes to Single-Cell Analysis

  • Significant progress has been made in the effort to overcome the technical challenges inherent in measuring biological phenomena in single cells. This progress has been made possible by applying approaches from very different fields of study.

    The goal in any biological study is to develop a method that allows you to “see” what is going on under physiological conditions without perturbing the system by the use of large labels or probes. With that in mind, Manlio Tassieri, Ph.D., Royal Academy of Engineering research fellow at the University of Glasgow, has developed a simple and noninvasive experimental procedure to measure the linear viscoelastic properties of cells, which he described at Select BioSciences’ “Single Cell Analysis” conference.

    How can rheology, the study of the flow of fluids, be used to develop models describing complex systems like cells? By applying rheology principles to the study of deformations occurring at micron length scales and with µL volumes, Dr. Tassieri and his group have contributed to the field of microrheology by moving their research focus from in vitro models to in vivo systems like living cells. In order to monitor the physiology in cells and get a sense of the underlying cytoskeleton, the team attached an antibody-coated 5 micron bead (anti-CD4) to a Jurkat cell resting on a coverslip.

    The team then tracks the movement of the bead in relation to the larger lymphocyte by video monitoring. In the test system, movement of the bead was influenced by the combined action of both the cell’s cytoskeletal activity and the thermal fluctuations of the fluid molecules within the media around the cell. For example, the response to a hypo-osmotic shock could be followed by tracking the bead.

    “Osmotic regulation is fundamental to homeostasis of the cell as documented in the literature. Using our microrheology method, we are able to show that when the cell is moved from iso- to hypo-osmotic conditions, the cell swells and then stiffens up to 300% of its normal rigidity,” explained Dr. Tassieri. “This rigidity is based on bundles of actin filaments realigning, leading to the re-organization of the cytoskeleton. Then over a period of minutes, the cell relaxes."

    Similarly, the response to the addition of blebbistatin, a drug that blocks muscle contraction via interference with the actin/myosin interaction, can be monitored in the same way as shown for osmotic shock. The approach taken by the Tassieri lab is robust and reproducible, when compared to assays using techniques such as magnetic tweezers, optical tweezers, and atomic force microscopy. Further, the method has the advantage of revealing changes over a wide range of frequencies to a high level of accuracy.

    Based on its simplicity and label-free nature, this application of microrheology could prove to be a valuable addition to studies that address cellular physiology under different pathological states.

  • Spectroscopic Analysis

    Click Image To Enlarge +
    University of Manchester researchers are utilizing IR spectroscopy for the analysis of cancer stem cell subpopulations. Infrared hyperspectral image of cancer stem cells (left) and a second derivative infrared spectrum (red) and normal cancer cells (right) with the second derivative infrared spectrum (yellow).

    Scientists at the University of Manchester, led by Peter Gardner, Ph.D., reader in the School of Chemistry and Analytical Sciences, are utilizing infrared (IR) spectroscopy for the analysis of cancer stem cell subpopulations. The application of IR spectroscopy is a well-established methodology in analytical labs for detection of impurities in chemical substances.

    “IR spectroscopy as applied to the complexity of single cells is a problem that must be approached with a different mindset. You need to look at the spectral patterns not as a series of peaks assigned to different molecules, but rather look at the whole pattern like a fingerprint,” explained Dr. Gardner.

    “A major breakthrough for us came a couple of years ago when we overcame the problem of excessive spectral distortion due to scattering, in particular due to a phenomenon known as resonant Mie scattering (RMieS). Scattering had more to do with the morphology of the cells than the biochemistry. That is, rounded cells showed high scattering, flattened cells showed less. We developed scattering correction algorithms, which meant that infrared spectroscopy can now robustly be used to study single cells.”

    One project in the Gardner lab is the study of cancer stem cells, a subpopulation of cells in solid tumor tissue that are more resistant to chemotherapy and believed to be responsible for cancer recurrence. As a model system, the lab uses a renal cell carcinoma cell line (2245R) where the stem-like cells of interest make up less than 5% of the entire population. In the absence of specific cell markers, the team has exploited the observation that these stem-like cells overexpress the multidrug-resistance (MDR) pump.

    Using a Hoechst dye efflux assay, the subpopulation of stem cells can be FACS sorted based on their lack of cytoplasmic dye and cytospun onto slides for spectral analysis using an imaging infrared spectrometer. IR spectroscopy signatures can be captured from thousands of cells in a rapid and reproducible data output. The spectral signatures can reliably differentiate cell populations—for example, differentiate between cells that overexpress the MDR pump from those that don’t.

    The spectral contrast comes from the cell makeup and is not dependent on stains or dyes. Routinely, it is possible to overlay an optical image of the cells with their IR spectral image so that each cell and the associated spectrum can be spatially located. The IR spectral signature, corrected for the scattering artifacts, registers only the absorption events that provide information about the cellular chemistry including glycogen levels as well as lipid and phosphate profiles.

  • Signaling

    Signal transduction based on a ligand binding to a receptor on the cell membrane and the subsequent cascade of phosphorylation that ultimately leads to gene expression in the nucleus that happens over a period of minutes is well understood. But what about the early events, the subsecond molecular processes that occur when the ligand binds the receptor?

    To capture these events, Jonathan West, Ph.D., and Ya-Yu Chiang, Ph.D., of the miniaturization group at ISAS, Dortmund, Germany, have developed a microfluidic system for the rapid treatment of single cells. The key is to use a two-step deterministic lateral displacement (DLD) technique that “bumps” cells from one laminar stream into a stream for receptor stimulation with ligand molecules and subsequently bump them into another stream for reaction quenching.

    The incubation time is precisely defined by the cell speed and the distance between the DLD switching elements. This design avoids the need to mix the ligand with the cells as that process takes too long (seconds) to effectively capture the initial events.


Add a comment

  • You must be signed in to perform this action.
    Click here to Login or Register for free.
    You will be taken back to your selected item after Login/Registration.

Related content

Jobs

GEN Jobs powered by HireLifeScience.com connects you directly to employers in pharma, biotech, and the life sciences. View 40 to 50 fresh job postings daily or search for employment opportunities including those in R&D, clinical research, QA/QC, biomanufacturing, and regulatory affairs.
 Searching...
More »

GEN Poll

More » Poll Results »

Should the CDC Director Resign?

Do you think the CDC chief should resign?