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

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

Two key issues for the pharmaceutical industry include accuracy of predicting in vivo drug interactions, including toxicity, and the time/cost of drug development. According to a 2007 report in Archives of Internal Medicine, the number of serious adverse and fatal drug events in the U.S., as reported to the FDA, increased threefold between 1998 and 2005.

One of the many advantages of 3-D cell culture is its ability to more accurately replicate physiological behavior, its proponents claim. Additional benefits are just beginning to be understood and utilized to enhance drug discovery, as well as bioprocess applications. Stem cells are also providing new opportunities in both of these areas.

When researchers in the laboratory of Robin Felder, Ph.D., at the University of Virginia, attempted to grow human renal proximal tubule cells on various 3-D technologies, they had problems.

“These are the leading cells in the kidney and have a definitive 3-D orientation,” noted Dr. Felder, professor of pathology and associate director of clinical chemistry. “If grown on a petri dish, it looks like a fried egg on a plate. It’s biologically confused.

“We decided to develop a microcarrier to get the cells oriented in the right polarity,” added Dr. Felder, who spoke on 3-D cell culture technology last month at CHI’s “Bioprocessing Summit” in Cambridge, MA.

Dr. Felder is also a cofounder and chairman of the board at Global Cell Solutions, which manufactures GEM™ (Global Eukaryotic Microcarrier). GEM contains alginate, a long, unbranched polysaccharide derived from brown seaweed, that acts like a “big electro-negative well that holds ions and molecules and allows the GEM to have a big sugar presence,” explained Brad Justice, director of research at Global Cell Solutions.

The Z® RP cell- and tissue-culturing system establishes a unique technological platform for fast and controlled expansion of adherent cells.

Different Technology

Justice said that this differs from previous 3-D technology that was based around proteins. The polysaccharide component seems to be important to the cells. Recent studies have also shown that alginate is biochemically sufficient to help maintain embryonic stem cells in an undifferentiated state.

Other advantages to GEM include the  fact that it is optically clear and allows the incorporation of magnetic particles. This enables automated cell manipulation by an externally applied magnetic field and prevents cell damage and debris. “The magnets are essential to automation, to spatially control the cells’ location, which is required in a system with no human intervention,” explained Justice.

The main cells currently being used with GEM include: HEK293, CHO, and HeLa and, recently, a large variety of stem cells. In addition, GEM allows users to grow numerous cancer cell lines, neural cells, epithelial cells, and various primary cells right from human or animal sources by engineering certain coatings to improve cell growth, such as selective biomimetic coatings, antibodies, and various growth factors.

Automation Is Key

One of the challenges in bringing microcarriers into research is a lack of easy-to-use systems and small volume containers. This is what the BioLevitator™ was designed to address. Codeveloped by Global Cell Solutions and Hamilton, this unit is a compact benchtop incubator and bioreactor hybrid for handling four independent, high-density 3-D cell cultures. Engineered to magnetically maneuver the GEM, it facilitates nutrient and gas exchange, providing good growth rates and relevant phenotypes, said Justice.

“It also gives us absolute control over the inoculation culture conditions,” he added.

Up to four BioLevitators can be integrated on a liquid-handling platform to create the 3-D CellHost™, which is based on Hamilton’s STAR platform, and is intended for various applications including drug discovery.

“Cell-based assays are now part of all stages of drug discovery so that cell supply has become a major bottleneck,” said Clara Cavelier, Ph.D., senior product manager, cell biology at Hamilton Robotics. “Stem cells may provide a virtually unlimited source of specialized cells to all stages of the drug discovery process, including target discovery, lead identification, and validation.”

The 3-D CellHost permits scientists to take cryopreserved cells, automatically retrieve them from an automated freezer, and automatically introduce them directly into the system, according to Dr. Felder, who says that the system combines biorepository function, cell culture, and analytical technology.

Recent research by Life and Brain demonstrates that this system facilitates human ES cell derived neural stem cell growth, supports undifferentiated cell growth, and maintains morphological characteristics, noted Dr. Cavelier, who gave a presentation on 3-D cell culture at the SBS annual conference in Lille, France, earlier this year.

“Cell culture automation eliminates variability and provides immediate and significant improvements in downstream cell-based assays, i.e., cell-based HTS,” she added. “The big hope is that stem cells will provide better models for drug discovery.”

Characterization of Hepatoma Cells

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.

Fluorescently labeled HeLa cells cover a collection of GEMs. Cell membranes are labeled green and the nuclei are blue.

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 (, 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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