Expanding Human Neurons
Students under the direction of Manuel Carrondo, Ph.D., at the Animal Cell Technology Lab based at the Instituto de Tecnologia Quimica e Biologica in Portugal (tca.itqb.unl.pt), have developed an efficient, scalable bioprocess for the expansion of human neurons. Undifferentiated NTera2 cells (human embryonal carcinoma stem cells) were expanded as 3-D aggregates in stirred bioreactors developed in Dr. Carrondo’s lab several years ago.
“We can keep the enzymatic activity for much longer—for over three weeks under those conditions. We’ve been able to show we can study metabolic activities under different insults (e.g., low oxygen) much better than under 2-D cell conditions,” said Dr. Carrondo.
In addition, characterization of the expanded cell population shows that the NT2 cells maintained their stem cell characteristics.
The neuronal differentiation step was done by addition of retinoic acid and its efficiency evaluated. The bioreactor process enhanced the differentiation efficiency 10-fold and reduced the differentiation time by 30%, when compared to other methods using static conditions.
“These cells are good models and express a lot of markers of major neurons in the CNS,” explained Margarida Serra, a Ph.D. student. “They can be used for in vitro toxicology, cell therapy, and drug screening applications.”
A key issue with embryonic stem cells is being able to grow them without differentiation.
“Most companies grow them in 2-D with robotic systems,” said Dr. Carrondo. “What we’re trying to improve is the expansion of undifferentiated stem cells to a much larger extent in 3-D systems without differentiation. The second step is once they’ve been growing under 3-D conditions, you want to direct them in just one direction as opposed to a random type of differentiation.”
The system helps to ensure standardization because the researchers were able to show they were able to exactly differentiate almost 90% of the cells in 3-D, whereas in 2-D the value would be only 20%, continues Dr. Carrondo.
The study demonstrated that through systems biology, by sampling the liquid component of the bioreactor, the researchers may interpret what is going on inside the 3-D aggregate of cells.
“This allows us to produce an enormous amount of data that indicates what’s happening, for example, when you have low oxygen levels or when using metabolic treatment,” according to Dr. Carrondo. “Those issues could make better tools for improved basic science, but also for more pragmatic and preclinical assessment of new drugs.”