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Oct. 5, 1999: If you've ever seen a pile of ivy that has taken the shape of an old barn that it has overgrown, you've seen the principle that researchers are following in trying to grow replacement parts for bodies. In research partly sponsored by NASA, scientists at the Massachusetts Institute of Technology have reported advances in characterizing the structural and electrical properties of heart tissue, and they've defined key parameters for growing the tissues.
Their results are reported in the August issue of the American Journal of Physiology - Heart and Circulatory Physiology and the September issue of Biotechnology and Bioengineering.
The work is led by Dr. Lisa Freed, a principal research scientist in the Harvard-MIT Division of Health Sciences and Technology, working with Dr. Gordana Vunjak-Novakovic and other colleagues at MIT, Harvard Medical School, Boston University, and Brigham and Women's Hospital.
Their work is supported by NASA's Biotechnology Cell Science Program, directed by Dr. Neal Pellis at NASA's Johnson Space Center. the NASA program involves more than 100 scientists, engineers, and support personnel around the nation. A series of experiments has been carried out aboard the Space Shuttle and Russia's Mir space station, and soon will be expanded aboard the International Space Station. The Cell Biology Program is managed by NASA's Microgravity Research Program at Marshall Space Flight Center in Huntsville, Ala.
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The Bioreactor was developed by NASA to simulate the weightless environment of space by putting cells in a growth medium that constantly rotates and keeps the cells in endless free-fall.
For many people, culturing cells means putting some small number into nutrient media in a dish or a tube and letting them grow. However, this kind of approach does not provide the culture environment that supports tissue assemblies. Without a proper 3-D assembly, epithelial cells (the basic cells that differentiate tissue into specific organ functions) lack the proper clues for growing into the variety of cells that make up a particular tissue.
In a rotating Bioreactor, the cells can be fooled into thinking they are in a body. With a plastic lattice to help direct their growth, cells can be encouraged to grow in predefined shapes, just as the vine-covered barn gives shape to vines.
Between September 1996 and January 1997, Freed and Vunjak-Novakovic with NASA colleagues achieved the first such results when they grew cartilage aboard the Space Station Mir in the first tissue-engineering experiment in space. They published their results in the December 1997 issue of the Proceedings of the National Academy of Sciences. This followed Freed's first successful experiment in engineering heart tissue: the cells she had "seeded" on a three-dimensional scaffold outside a living body began beating as one. "It was my most awesome laboratory moment ever. No one had ever done this before," said Freed. That work was completed in 1994 and published in 1997.
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The MIT work is key to engineering three-dimensional cardiac tissue that could eventually be used to repair damaged heart tissue inside the body, test new drugs, and study general cardiac tissue development and function. Although it could theoretically lead to the creation of an entire heart, the researchers stress that substantial problems must be solved before that could happen. For example, while the current constructs resemble heart muscle, they lack blood vessels. "We've developed one component, but that is only the first step," Freed said. The MIT approach involves seeding cardiac cells onto a 3D polymer scaffold that slowly biodegrades as the cells develop into a full tissue. The researchers have used the same technique to grow other tissues.
The cardiac cells are cultivated on scaffolds 5 mm in diameter by 2 mm thick. The cell/scaffold constructs are placed in a rotating bioreactor that supplies the cells with nutrients and gases and removes wastes.
"The bioreactor is a kind of microenvironment that gives cells the signals they would ordinarily see in the body," said Vunjak-Novakovic. "This overall system allows us to study specific effects of the cells, scaffold, and regulatory signals on tissue development and function," Dr. Freed said.
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Left:
A ground-test model of the NASA Bioreactor shows the key element,
a rotating plastic cylinder enclosing a tubular membrane that
infuses growth media and oxygen and removes wastes. Credit: NASA/Johnson
Space Center.In Biotechnology and Bioengineering, the team described how parameters like cell density, cell source (neonatal rat or chick embryo), and different cultivation conditions affect tissue growth. This work included the NASA Bioreactor and conventional spinner flasks.
"We've identified a set of conditions that so far appear to be best for cardiac tissue engineering," Vunjak-Novakovic said.
Work continues.
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"The type of mammary cells we are growing
comes from an individual susceptible to breast cancer, and that
susceptibility is likely driven by damage caused by ionizing
radiation. Space exploration will involve slightly increased
exposures of crew members to radiation, so what we learn from
these cells could help help justify methods of female crew selection,
and help manage breast cancer in the national population at the
same time.""There are substantial problems that must be addressed before we could use these tissues for, say, repair of heart defects inside the body," Freed said.
In addition, constructs must be bigger, stronger, and made of human rather than animal cells that have been modified so they will not be rejected by a recipient.
The first successful experiment five years ago "showed that cardiac tissue engineering was possible," Vunjak-Novakovic said. The current papers are the first to quantitatively characterize tissue properties.
"They're really the beginning," she concluded.
"Cardiac muscle tissue engineering:
toward an in vitro model for electrophysiological studies."
American
Journal of Physiology, Heart and Circulatory Physiology. Vol.
277, Issue 2, H433-H444, August 1999.
The objective of this study was to establish a three-dimensional (3-D) in vitro model system of cardiac muscle for electrophysiological studies. Primary neonatal rat ventricular cells containing lower or higher fractions of cardiac myocytes were cultured on polymeric scaffolds in bioreactors to form regular or enriched cardiac muscle constructs, respectively. After 1 wk, all constructs contained a peripheral tissue-like region (50-70 µm thick) in which differentiated cardiac myocytes were organized in multiple layers in a 3-D configuration. Indexes of cell size (protein/DNA) and metabolic activity (tetrazolium conversion/DNA) were similar for constructs and neonatal rat ventricles. Electrophysiological studies conducted using a linear array of extracellular electrodes showed that the peripheral region of constructs exhibited relatively homogeneous electrical properties and sustained macroscopically continuous impulse propagation on a centimeter-size scale. Electrophysiological properties of enriched constructs were superior to those of regular constructs but inferior o those of native ventricles. These results demonstrate that 3-D cardiac muscle constructs can be engineered with cardiac-specific structural and electrophysiological properties and used for in vitro impulse propagation studies.
"Cardiac Tissue Engineering: cell seeding, cultivation parameters, and tissue construct characterization." Biotechnology and Bioengineering, 64: 580-589, September 1999. R. Carrier, M. Papadaki, Maria Rupnick, F. J. Schoen, N. Bursac, Robert S. Langer, L. E. Freed, G. Vunjak-Novakovic.