## Neurociencia | Neuroscience

#### Neuroscience

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### B

#### La arrogancia como estilo en la práctica de la medicina

Esa vieja y terrible enfermedad "Broncemia". Una magistral conferencia del Dr. Francisco Occihuzzi pronunciada en TED Córdoba. La arrogancia como estilo en la práctica de la medicina. Un patético modo de alejarse del dolor de la gente y del afecto de sus colegas.

Seguir leyendo en el sitio web http://kwfoundation.org

 Palabra(s) clave: ArroganciaBroncemia

### Bugs as Drugs: Seeking the Microbial Secret to Health

Our body is, in essence, more ecosystem than organism.

The human body teems with trillions of microbes — bacteria, viruses and fungi — and at any moment, we may be carrying between one and three pounds of these micro-hitchhikers in colonies on our skin, groin, mouths, and sinuses. By far, however, the gut microbiome ecosystem — the largest and most complex — is the one that has both academic researchers and pharmaceutical companies hooked.

The reason? Our gut bugs may be the new frontier of a billion-dollar bioceutical industry.

#### 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 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 | PDF

REDRAWN FROM "NARROW STRAIGHTS," THE SCIENTIST, JULY 2013

CellSqueeze 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 Harvard Medical School who cofounded SQZ Biotechnologies withRobert 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

• 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.

#### 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 | PDF
REDRAWN 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 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.

 Palabra(s) clave: Cell TransfectionMolecular DeliveryTechnologies

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