Researchers from Rice University and the M.D. Anderson Cancer Center at the University of Texas have developed a new way to coax cells to assemble into 3-D formations while in culture. Traditional 2-D cell cultures are an unnatural environment for cell growth, resulting in changes to gene expression, signaling, and morphology in the cultured cells. To avoid these unintended effects, researchers have developed 3-D cell culture techniques that use protein-based gels and rotational and agitation-based bioreactors. However, a straightforward, easily applied 3-D culture technique has eluded researchers until now.
"There's a big push right now to find ways to grow cells in 3-D because the body is 3-D, and cultures that more closely resemble native tissue are expected to provide better results for preclinical drug tests," Tom Killian, associate professor of physics at Rice University, and co-author on the study, said in a press release. "If you could improve the accuracy of early drug screenings by just 10%, it's estimated you could save as much as $100 million per drug."
To address the problems surrounding current 3-D culturing methods, the Rice–M.D. Anderson team developed a bioassembler. Their bioassembler system uses magnetic forces—delivered with varying sized magnetic rings—to levitate developing cells and encourage culture growth into 3-D shapes. The levitation process is based on a bioinorganic hydrogel, composed of three parts a bacteriophage, magnetic iron oxide, and gold nanoparticles that self-assemble into hydrogels. The bacteriophage used by the team is M13-derived and displays the ligand peptide RGD-4C to target the gold nanoparticles and magnetic iron oxide. This magnetic ring-based technology is compatible with all standard culturing techniques and thus can be used in most laboratories.
To construct the culturing system, the researchers added the nanoparticles and the bacteriophage to the hydrogel containing the cells for culture. Due to the ligand peptide expressed by the bacteriophage, it targeted the nanoparticles. When the bacteriophage infected the cells, it then transported the nanoparticles into the cell. The researchers then washed away the gel and seeded the nanoparticle-loaded cells in a Petri dish filled with a substrate that promoted cell growth. Once the cells were loaded with the nanoparticles, which would react to the magnetic forces, the researchers placed a magnetic ring on top of the Petri dish.
To assess cell growth through this technique, the researchers visually and quantitatively monitored the formation rate, size, and viability of human glioblastoma cells over a period of eight days. They monitored the cells using red fluorescent mcherry-tagged proteins.
After 24 hours, a cohesive multicellular assembly became suspended in the liquid, according to the researchers. A spheroid shape, which reached a maximum diameter of 1mm took form between the next 72 hours and 192 hours. The researchers cultured cells that they hoped would develop a glioblastoma, a cancerous tumor. After testing for proteins that glioblastomas produce in vitro, the researchers found the appropriate proteins expressed in the 3-D culture, but not in a comparable 2-D culture. The expression of the glioblastoma proteins makes the 3-D culture appropriate for studying these cancerous tumors. The lack of the proteins in the 2-D culture indicates that it would not be a valid mechanism for studying cancer.
"The beauty of this method is that it allows natural cell-cell interactions to drive assembly of 3-D microtissue structures,” said Robert Raphael, an associate professor in the department of bioengineering at Rice. “The method is fairly simple and should be a good point of entry in 3-D cell culturing for any lab that's interested in drug discovery, stem cell biology, regenerative medicine or biotechnology."
3-D tissue cultures have promising applications for studies of diseases, such as cancer, which behave differently in vitro. “For cancer research, the ’invisible scaffold’ created by the magnetic field goes beyond its potential for producing cell cultures that are more reminiscent of real tumors,” said Wadih Arap, professor at the M.D. Anderson Cancer center and co-author of the recent paper. "A logical next step for us will be to use this additional magnetic property in targeted ways to explore possible applications in the imaging and treatment of tumors.”
The research was funded in part by M.D. Anderson's Odyssey Scholar Program, the Department of Defense, the National Science Foundation, and the National Cancer Institute. The University of Texas has filed a patent for the technology on behalf of the researchers. The paper, “Three-dimensional tissue culture based on magnetic cell levitation,” was published Mar. 14 in Nature Nanotechnology.