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Sep 15, 2009 (Vol. 29, No. 16)

3-D Cell Culture for Discovery & Bioprocess

Approach Gaining in Popularity Due to Its Ability to Mimic an In Vivo Growth Environment

  • Characterization of Hepatoma Cells

    Click Image To Enlarge +
    Fluorescently labeled HeLa cells cover a collection of GEMs. Cell membranes are labeled green and the nuclei are blue.

    Human hepatoma cells may provide an excellent model to predict toxic effects of pharmaceuticals on the human body, avoiding animal testing. Researchers in Germany compared 2-D and 3-D cell cultures of hepatoma cells (HepG2) for their xenobiotic metabolizing function, determined by measuring EROD, a biomarker that indicates a liver cell is digesting a toxin through the cytochrome pathway.

    “Measuring the amount of this biomarker should directly correlate with the toxic effect of a substance incubated with the liver cell,” explained Hans Hoffmeister, Ph.D., CEO, Zellwerk.

    Normally, liver cells do not activate that pathway, but the 3-D culture induces this detoxification ability and, therefore, makes hepatoma cell lines a candidate for drug testing (currently only primary hepatic cells have been used).

    The cells were grown on Zellwerk’s Sponceram® macroporous ceramic carriers, which have large surface areas (up to several hundred square meters), in a 500 mL 3-D bioreactor (Z® RP bioreactor; distributed in North America by Glen Mills). This setup, which allowed high cell densities, can also be adapted to each cell’s specific needs for anchoring moieties, pore size, and chemical/structural microenvironment.

    “Hepatoma cells, as well as many other types, arrange in a 3-D fashion including embedding themselves in extracellular matrix and develop characteristics of primary cells, i.e., change their metabolism from an undifferentiated cell line to a metabolism close to a primary cell,” said Dr. Hoffmeister, who discussed his company’s work on hepatoma cells at the “ESACT” conference in Dublin in June.

    The study data show that maximum EROD activity on the Sponceram was 2.23-fold on day 12 versus the activity of monolayer cells on day 7 of cultivation. This demonstrated that 3-D cultivation resulted in improved functional characteristics versus monolayer culture.

    All cell types that are anchorage dependent can be used with the Sponceram—producer cell lines for biopharmaceutical production, stem cells for cell therapy, and primary human cells for tissue engineering, according to Dr. Hoffmeister. He added that potential future applications for this carrier include the manufacture of large tissue pieces for patient-specific bone/cartilage for arthritis implants.

  • 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.”

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