Ecole Polytechnique de Lausanne. Switzerland: Scientists at EPFL have developed a new lab-on-a-chip
technique to analyze single cells from entire population. The new
method, which uses beads and microfluidics can change the way we study
mixed populations of cells, such as those of tumors.
Individual cells in a population, e.g. a tumor, can
vary greatly in terms of biochemistry and function. In order to fully
understand and address this variability, it is necessary to profile the
properties of each individual cell, such as the interactions between its
DNA, RNA and proteins. This has always been a challenge due to the
technical and physical limitations associated with the fragility and
small size of cells, as well as the large variety yet low amount of
biomolecules they contain. EPFL scientists have now combined, for the
first time, affinity beads with microfluidics to produce an integrated,
highly sensitive method for studying single cells that could be soon
used in clinical diagnostics. The pioneering work is published in the
journal Small.
There are two major obstacles for
efficient single-cell analysis: first, the large number of different
types of low quantity of biomolecules that have to be investigated in
each cell. Second, the sheer number of cells that have to be processed,
especially when trying to detect rare changes, which occur only in a few
cells of a large population.
The lab of Horst Vogel at EPFL was
able to overcome these hurdles by combining several micro-technologies:
Micrometer- to submicrometer-sized affinity beads transferred into
cells, and extraction of these beads from individual cells in a
microfluidic channel. In the technique, developed by PhD student Michael
Werner, the target molecule inside a single cell is captured on
(sub)micrometer-sized silicon beads, which have previously been coated
with capture agents, e.g. antibodies, that can bind specifically to the
target biomolecule in the cell.
When taken up by a cell, the bead
is first internalized in a closed intracellular cage called the
phagosome. This creates a problem, as the phagosome keeps the bead from
finding its target biomolecule inside the cell. In order to release the
bead from the phagosome, the researchers used a photochemical trick.
Along with the beads, they also incubated the cells with photosensitive
molecules, which became incorporated into the phagosome’s membrane.
When
light is shone on the cells, the photosensitive molecules disrupt the
phagosome’s membranes. With the membranes ruptured, the beads are now
released inside the cell, where they capture and extract all of the
target molecules from the cell’s cytoplasm with high specificity.
Next,
the bead-containing cells are put through a microfluidic chip. This is a
small device designed specifically to control the flow of tiny volumes
of fluids across a network of small channels (100 μm in width) etched
into glass slides. The channels in this study are so narrow that cells
can only pass through one at a time. The cells pass through a point in
the channel where a “laser tweezer” (a highly focused laser beam)
catches individual cells by interacting with their internalized beads.
The trapped cells are then lysed, leaving only the beads with the bound
target biomolecules in the laser trap. The beads are then analyzed
directly inside the microfluidic device.
In the EPFL study, the
scientists explored a number of different cell types, providing a proof
of concept for their new single-cell analysis method. The team is now
working with the Centre hospitalier universitaire vaudois (CHUV) in
Lausanne to test their assay on cancer cells from actual tumors, which
show enormous variability in signaling profiles between individual
cells. “We hope that characterizing these variations will improve the
treatment of the disease on an individual basis”, says Horst Vogel.
Reference
Werner M, Palankar R, Arm L, Hovius R, Vogel H. Microfluidic Single-Cell Analysis with Affinity Beads. Small DOI: 10.1002/smll.201402650