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Live Cell Extractions

Nanostraws that collect specimens from cells without killing them allow for repeated sampling.

Oct 1, 2017
Ruth Williams

EXTRACTION WITHOUT KILLING: Cells are cultured atop a polycarbonate membrane perforated in spots by vertical aluminum oxide nanostraws. At defined locations, where the membrane has been etched away lithographically, the nanostraws protrude from the membrane and contact the cells. A brief electric voltage is passed across the nanostraws, causing temporary perforations in the cell membrane. This allows small volumes of cytoplasm to diffuse into the nanostraws for collection in the reservoir of extraction buffer below the polycarbonate membrane.
See the full infographic: WEB 
© GEORGE RETSECK

Analyzing cells en masse provides a general idea of the happenings within a given cell type, but misses the subtle yet significant variations between individual cells—variations that may result in different responses to developmental signals, drugs, and other factors.

To better explore the inherent heterogeneity of cell populations, “many people are trying to do single-cell analyses,” says Orane Guillaume-Gentil of the Swiss Federal Institute of Technology (ETH). But, she adds, the approaches are limited. “You have to kill the cells, so you cannot see anything dynamic, and you also lose the [spatial] context of the cells.”

The problem, agrees Nicholas Melosh of Stanford University, is that “you want to know what a cell is, but [current single-cell approaches] tell you what it was.”

Researchers are therefore developing nondisruptive techniques to enable the sampling of live cells without having to isolate or destroy them. Melosh recently devised a nanostraw extraction (NEX) technique that is the latest addition to this toolkit.

While previous techniques, from Guillaume-Gentil and others, used nanopipettes, nanotubes, or similar sampling devices that penetrate cells from above, in Melosh’s approach, cells are grown on a polycarbonate membrane containing alumina nanostraws, which protrude through the membrane from below in a defined location. The nanostraws do not pierce the cells under normal conditions, but an electric current passed through the straws briefly opens pores in the cell membrane, allowing contents to diffuse into the straws for collection.

By having a nanostraw-dotted surface rather than a single sampling device, Melosh’s approach has the potential to become high-throughput, says Guillaume-Gentil, who was not involved in the study.
Melosh’s team used NEX to analyze mRNAs and fluorescent proteins expressed in single cells or small groups of cells for a period of several days.

While “other approaches have shown good cell viability and the potential to do such [dynamic studies],” says Guillaume-Gentil, Melosh and colleagues “are the first ones that really showed that they can [do it]. It was an important proof of principle.” (PNAS, 114:E1866-74, 2017)

APPROACH HOW IT WORKS DNA MUTATION ANALYSIS TRANSCRIPTOMICS PROTEIN ANALYSIS
Current single-cell analyses Single cells are isolated and lysed and their contents analyzed by sequencing or proteomics methods. Commercially available kits for whole or partial genome sequencing Commercially available kits for RNA sequencing A number of techniques exist, some commercially available, that analyze proteins via antibody binding, mass spectrometry, or other means.
NEX Live cells in culture have a small portion of their cytoplasmic contents removed for analysis. The cell is minimally disturbed, survives, and can be repeatedly sampled. Not yet tested Messenger RNAs from small groups of cells can be analyzed by sequencing, but single-cell analysis is not yet possible. Specific fluorescent proteins can be monitored over several days in single cells or small groups of cells.
 

 

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