2. Still others are working on completely new approaches for delivering not only nucleic acids but also
additional types of materials such as small molecules and proteins. Here, The Scientist brings you the
lowdown on some of the newest transfection and molecular-delivery products and methods.
Exo-Fect
System Biosciences
systembio.com
EXO-FECT: Isolated exosomes are transfected with
nucleic acids of interest using the nonliposomal transfection reagent, Exo-Fect. Transfected exosomes
are then added to cells, which internalize the vesicles along with their cargo.
See full infographic: JPG | PDFTHE SCIENTIST STAFFExo-Fect is a nonliposomal transfection reagent that
initially delivers nucleic acids, including plasmid DNA, mRNA, microRNA, and siRNA, directly into isolated
exosomes—naturally occurring extracellular vesicles that are shed from most cell types and are present
in most bodily fluids. Exosomes are thought to function in intercellular communication; after they are
released from cells, exosomes can fuse with distant cells or become internalized through endocytosis.
Transfected exosomes can therefore serve as delivery vehicles for nucleic acids of interest. “You add the
exosomes to cells, and they will deliver that cargo that you put in,” says Travis Antes, senior director of
product development at System Biosciences.
To get started, users will first need to isolate exosomes, which can be accomplished by
ultracentrifugation of cell-culture media or bodily fluids, or by using a commercial exosome-isolation kit
available from multiple vendors including System Biosciences, Life Technologies, and others.
Alternatively, researchers can purchase pre-isolated exosomes from System Biosciences. The
transfection protocol is relatively straightforward: Mix isolated exosomes with the nucleic acid to be
delivered and the Exo-Fect reagent, heat at 37 °C for 10 minutes, and then chill on ice for half an hour.
Add a second reagent to precipitate the exosomes so that they can be separated from the transfection
reagent and any untransfected nucleic acid. The entire procedure takes about 45 minutes to complete
and requires roughly one million exosomes per transfection reaction, which should be sufficient for
exosome-mediated delivery of molecules to cells in two wells of a six-well culture plate.
3. Benefits
Exosome-mediated delivery is nontoxic, Antes says. “Once we add the exosomes onto cells,
there’s zero [cell] death.”
Can be used to deliver mixtures of different nucleic acid types
The Exo-Fect transfection kit comes with a fluorescently labeled nontargeting siRNA that can be
used to monitor the effectiveness of a transfection and subsequent delivery into cells.
Challenges
Depending on the method used, exosome isolation can be time-consuming and labor-intensive.
The product was only released in April 2014, so it remains relatively untested by researchers.
The fetal bovine serum (FBS) that is typically added to cell culture media is chock-full of cow
exosomes that can interfere with exosome-mediated delivery. The company recommends users
grow their cells in media with exosome-depleted FBS.
Cost
Kit costs range from $195 for 10 transfection reactions to $350 for 20 reactions. Exosome isolation kits
start at $288, and a 50 mL bottle of exosome-depleted FBS sells for $153. The company also provides
pre-isolated exosomes from a variety of sources starting at about $350 for a vial containing roughly one
million exosomes.
CellSqueeze
SQZ Biotechnologies
sqzbiotech.com
CELLSQUEEZE: The rectangular microfluidic chip
contains a series of parallel channels, each with at least one narrow constriction designed to be smaller
4. than the diameter of a cell. As the cells squeeze through the constrictions, pores form transiently in the
plasma membrane, allowing extracellular molecules to enter the cytoplasm by diffusion. The cell
membrane then reseals within minutes.
See full infographic: JPG | PDFREDRAWN FROM "NARROW STRAIGHTS," THE SCIENTIST, JULY
2013CellSqueeze is a microfluidic system released to the MARKET in 2013 that can deliver a variety of
materials, including siRNA, drugs, proteins, or nanoparticles, into virtually any cell type. (See “Narrow
Straits,” The Scientist, July 2013.) The system uses a rectangular microfluidic chip containing a series of
75 parallel channels, each of which is 30 microns in diameter and contains at least one narrow
constriction designed to be smaller than the diameter of a cell.
As the cells squeeze through the constrictions, transient pores form in the plasma membrane, allowing
extracellular molecules to enter the cytoplasm by diffusion. The cell membrane then reseals within
minutes. Disrupting the cell membrane in this way “doesn’t seem to have any long-term side effects on
the cells,” says Armon Sharei, a postdoctoral fellow at HarvardMedical School who cofounded SQZ
Biotechnologies with Robert Langer and Klavs Jensen of MIT. “So it looks like we just open up their
membrane and they repair it after the stuff is in, and they don’t think anything of it,” adds Sharei.
The system has a pressure regulator that allows control of the speed with which the cells flow through
the channels, and a pair of reservoirs that sit atop the chip and interface with its inlet and outlet holes.
Users simply add their material to be transfected to a sample of cells in solution, deposit the mixture
into the one of the interchangeable reservoirs, and apply pressure to begin pumping the sample through
the device. Cells that have passed through the chip collect in the opposite reservoir, where they can be
retrieved. It only takes about 5 seconds for a sample to flow through the system, Sharei says.
Benefits
Easy to use and very fast, says user Morgane Griesbeck of the Ragon Institute of Massachusetts
General Hospital, MIT, and Harvard who used the system to introduce a recombinant protein
into a rare subset of human primary blood cells, “without stressing them too much, which is
something very difficult,” she adds.
Simple process that works well with a variety of cell types, including established cell lines,
primary immune cells, and embryonic stem cells
Can deliver a medley of materials simultaneously
The company OFFERS 16 different chip designs in which the length, width, and number of
constrictions per channel vary, so researchers can tweak a variety of parameters to try to get
the delivery that they desire.
Can reliably deliver molecules up to 2 MDa in size. “Bigger things probably get in too, but that’s
the biggest we’ve tested,” Sharei says.
Unlike conventional delivery strategies, the process doesn’t involve proprietary buffers or
delivery vectors that might be toxic to cells.
Challenges
5. The system is not currently suitable for delivering DNA and mRNA. “We know the mRNA and
DNA get inside cells, but once they’re inside, something prevents them from getting expressed,”
Sharei says. “We think we know what that is, and initial tests show that we may be able to get
around it.”
The reservoirs hold a maximum volume of 250 μL. Larger volumes can be processed in small
batches sequentially.
Requires two to three training sessions to learn how to use
The holder that clamps the reservoirs onto the chip will need to be replaced periodically
because it tends to loosen over time, causing leaks that can ruin experiments, Griesbeck says.
Cost
Chips sell for $50 apiece. A starter kit consisting of the pressure system plus two holder sets is
available for $3,000. Onsite training will set new users back about $800 to $1,000. The system is
commercially available only to “approved partners,” Sharei says. Prospective users will need to
consult with the company’s scientific team before they can gain access to the technology.
GOLD nanoparticle–mediated laser transfection (GNOME)
Leibniz University Hannover, Germany
GNOME: Cells are incubated with GOLD
nanoparticles (top); once the particles have settled on the cells, the molecule to be transfected is added
as gold particles adhere to the cell membrane (middle); and irradiation with very brief pulses of a weakly
focused green laser beam causes tiny holes to form in the cell membrane, allowing the diffusion of
extracellular molecules into the cytoplasm (bottom).
See full infographic: JPG | PDFREDRAWN FROM PLOS ONE, 8:E58604, 2013.Laser-based transfection
uses very short pulses of light to poke tiny holes in the cell membrane, allowing the diffusion of
extracellular molecules into the cytoplasm. The strategy has been used by laser specialists to deliver
different molecules into cells for at least a decade, but it is has traditionally been painstakingly slow and
low-throughput because the laser must be precisely focused on a cell with submicron resolution, one
cell at a time.
To speed up the process, Dag Heinemann, a postdoctoral fellow at the Laser Zentrum Hannover e.V. in
Germany, and his colleagues first incubate their cells with gold nanoparticles that are roughly 200 nm in
diameter. After about three hours, the particles settle onto the cells, and the researchers irradiate the
sample with very brief pulses of a weakly focused green laser beam with a diameter of about 90
6. microns. The irradiation is performed in an automated device that the researchers developed in-house,
complete with a microscope stage and software that automatically moves the culture plate around to
quickly irradiate all or parts of the sample.
Upon absorbing the light, electrons in the gold particles oscillate rapidly and heat up. What happens
next is not well understood, Heinemann says, but the end result is that the cells’ membranes become
perforated. Using the technique, Heinemann’s team delivered siRNA and effectively knocked down a
gene in a canine cancer cell line (PLoS One, 8:e58604, 2013). The team estimated that nearly 90 percent
of the cells were transfected, and more than 80 percent of the cells remained viable after the treatment.
Benefits
Very gentle. “We can achieve very high cell viabilities, which are typically above 90 percent even
with a sensitive cell type,” Heinemann says.
High-throughput. Heinemann says that an entire 96-well plate of cells can be processed in about
4–5 minutes.
Compatible with a variety of cell types and highly reproducible, “because the physical
mechanism stays the same and the cell itself is not actively involved in the mechanism,”
Heinemann says. Working in collaboration with researchers at the Hannover Medical School,
Heinemann says he has successfully transfected several different cell lines as well as primary
neurons, cardiomyocytes, and stem cells, which tend to be difficult to transfect using
established methods.
In addition to delivering siRNA, Heinemann has also used the method to deliver proteins, small
molecules, and synthetic oligonucleotides called morpholinos.
Challenges
Doesn’t work very well with plasmid DNA or other relatively large molecules. “We think the
plasmid is quite large for the type of openings we introduce with the particles,” Heinemann
says.
The GOLD particles eventually enter the cells in the process and could potentially alter cell
behavior. “But as much as we know, [the particles] are completely biologically inert and they do
not affect the cell afterward,” Heinemann says.
It’s still in the experimental stage at this point. Heinemann says he’s currently developing a user-friendly
prototype device that could be operated in a typical cell biology lab with a simple press
of a button. He says he hopes to have the system ready within a year.
Cost
None available. But Heinemann expects that his device will be competitive with sophisticated
electroporation systems, which typically retail for about $10,000 or more.
Tags
transfection, techniques, siRNA, nanoparticle, microfluidics, exosomes and cell & molecular biology
