Microfluidic cell culture offers many distinct advantages, not least that scientists can work with expensive rare cells, use less reagent, and in many cases control the environment with greater accuracy than in bulk cell culture, petri dishes, or multiwell plates. Samuel Forry, Ph.D., research chemist, National Institute of Standards and Technology (NIST), talks about technology developed at his institution.
“At NIST we’re very interested in fundamental measurements and the way measurement can improve biology. I think we have demonstrated a level of control and ability to measure the microenvironment around cells, which is revealed in sensitivities to partial gas pressures that may not have been appreciated,” notes Dr. Forry.
To accomplish this, Dr. Forry and his colleagues have fabricated the microchamber and microfluidic compartments out of gas-permeable material—poly(dimethylsiloxane)(PDMS)—and routed them near each other. “We can then allow diffusion through the material to give us control over the partial pressure in the culture chambers where the cells are being cultured. We’ve shown this for oxygen and for CO2.”
Essentially, they create stagnant conditions but are able to supply sufficient gases to keep the cells from disrupting the homeostasis in the environment. “We perfuse media past the cells but do it only intermittently,” he notes. “It turns out conventional cell culture media has enough salts, amino acids, and sugars to last for a pretty long time, but the gas partial pressures get out of whack quickly. We control the gas partial pressures directly through the material.”
The most common use, according to Dr. Forry, “is where you want to create a hypoxic environment or maintain 5% CO2. We can also create gradients across a microfluidic chamber to create systems where one side of our chamber has normal oxygen level or maybe 21% and the other side has hypoxic conditions and one can look at the way cells respond in the two different environments side by side.”
Rethinking Shear Sensitivity Stress
Shear sensitivity is an often-cited cause for bioreactor failure. Jeffrey J. Chalmers, Ph.D., professor, chemical & biomolecular engineering, Ohio State University, poses questions about the validity of this worry.
“Shear sensitivity is misused. It’s a bad term to start with. It’s used as an excuse when people don’t know why things aren’t working well,” says Dr. Chalmers, noting how the idea that cells could not be grown in suspension was held with equal faith 25 years ago and proved dreadfully wrong. For example, “When no surfactant was used to prevent cell adhesion to interfaces, people used to blame death like that onto mixing when it was due to other interactions. If the cells are in suspension, not attached to a microcarrier, they are pretty tough,” he insists.
No doubt it’s possible to have clones that are more susceptible to thermodynamic forces, he says, “but that’s not usually observed in the bioreactor because that’s a pretty gentle area. Downstream processing is where people can see problems.” Dr. Chalmers’ group has developed a device to help companies test clones to determine how sensitive they may be to specific dynamic forces.
Matrix-Free 3D Spheroid Technology
Eight to ten years ago, “we said we’ve got video cameras, we’ve got money, whole animal research will come to dominate,” says Mark DeCoster, Ph.D., associate professor, biomedical engineering, Louisiana Tech University (LTU). Since then budgetary constraints have changed the rosy picture. “It’s forced us to become more realistic. So while 2D cell culture won’t go away anytime soon, an intermediate 3D option is needed.”
“We have established a novel matrix-free 3D cell spheroid system that permits growth and maintenance of normal cells, stem cells, and cancer cells,” continues Dr. DeCoster. “In addition to processing of soluble drugs, we are also using our 3D system to evaluate bioprocessing of micro- and nanomaterials.
“We have measured binding and internalization of these materials as well as toxicity of nanomaterials. It is anticipated that 3D systems will provide new information for materials bioprocessing compared to traditional 2D cell culture systems due to differences in diffusion and cell-cell communication.”
His LTU lab concentrates on neuroscience. “Many brain tumors in the body have regions, a core that died, while the outside is growing. What’s quite satisfying to us and others too is that spheroid models recapitulate that; after they reach a certain size, the limits on glucose and oxygen’s ability to enter the spheroid cause it to have a necrotic core, yet the spheroid still grows because there is sufficient oxygen and glucose on the outside. Our system is excellent for observing these kinds of activity.”
He founded a startup—Nanogaia—to commercialize the technology. An important remaining challenge, he notes, is developing needed IT technology: “Storage has become incredibly cheap; the question is algorithms and software to turn the pretty pictures into meaningful things.”