Cell-Culturing System Advances Enhance Cell Growth Efficiency

Cell culture is widely used today in the production of various biologically active materials, such as viral vaccines, monoclonal antibodies, hormones, enzymes, and tumor-specific antigens. These items are produced by normal, transformed, and genetically engineered cells. The large-scale cultivation of specific cell lines is of major importance in the cost- effective manufacturing of many therapeutic proteins. >From laboratory benchtops to production floors, and even in outer space (see accompa

By | April 3, 1995

Cell culture is widely used today in the production of various biologically active materials, such as viral vaccines, monoclonal antibodies, hormones, enzymes, and tumor-specific antigens. These items are produced by normal, transformed, and genetically engineered cells. The large-scale cultivation of specific cell lines is of major importance in the cost- effective manufacturing of many therapeutic proteins.

>From laboratory benchtops to production floors, and even in outer space (see accompanying story), life sciences researchers require cell-culturing techniques that are more productive, easier to use, and more closely simulate in vivo situations. Yet traditional methods of producing these cell lines have left something to be desired, according to these scientists.

On the laboratory scale, anchorage-dependent cells, which must attach themselves to a substrate to grow and propagate, are often cultured in stationary T-flasks or roller bottles in batch-type cultures. These conventional processes are highly labor-intensive, typically requiring many growth vessels for the production of large amounts of cells. In addition, the cells must be removed, separated, and reinoculated whenever the medium is replenished.

Another well-established cell-culturing technique is the use of ascites mice. These animal models are injected with antibody-secreting cells, such as hybridomas, and the resulting monoclonal antibodies are removed in the ascites fluid. However, this practice presents a unique set of challenges. In addition to the logistics that must be worked out for housing, feeding, and caring for these animals, the fluids they produce generally contain an assortment of other proteins, peptides, and lipids that frequently cause considerable interference during the purification of the products.

But over the last few years, equipment manufacturers have responded with a variety of alternative cell-culture systems that improve upon the maintenance-intensive ascites mice and inefficient stationary-culture and roller-bottle methodologies.

The miniPERM, from Heraeus Instruments of South Plainfield, N.J., is a bioreactor-type device that simplifies the high- density cultivation of hybridomas and other cells, and facilitates the cost-efficient production of high-purity monoclonal antibodies and other extracellular products in suitable volumes for screening purposes.

This system consists of an autoclavable nutrient container and a disposable bioreactor. The latter is separated from the nutrient chamber by a semipermeable dialysis membrane that allows for the passage of nutrients and removal of metabolic wastes. Actively growing cells in the bioreactor may be easily separated from the nutrient container when nutrient charging is required, eliminating the need for multiple concentration steps, according to the company.

"The principal advantage of the miniPERM is that the cells are contained in that chamber at all times, even when the nutrient is spent and must be replaced," says Mary Gardineer, a Heraeus applications specialist. The user snaps off the bioreactor from the nutrient container, replenishes the medium, and rejoins the two components, Gardineer explains. During cultivation, the device is rotated in a CO2 incubator.

In general, the miniPERM is capable of achieving antibody concentrations up to 100 times higher than stationary cultures. Depending upon the characteristics of the hybridoma cell lines utilized, it's possible to obtain cell densities in excess of 107 cells per ml, and monoclonal antibodies of several mg per ml, claims Gardineer. Up to 160 mg of monoclonal antibodies may be produced in one to four weeks.

Laboratory bioreactors, with their wide variety of sizes and configurations, provide a closely controlled environment for cell-culture systems. Foster-City, Calif.-based Applikon Inc., as well as other suppliers, offer vessels ranging from 1 to 130 liters, with numerous agitation systems for mixing and distribution of nutrients. A multiport arrangement for the insertion of various types of sensors permits the monitoring of parameters such as pH, temperature, dissolved oxygen, CO2, and oxidation-reduction potential.

By growing insect cells in a bioreactor, David Clemm, a research scientist at San Diego-based Ligand Pharmaceuticals Inc., pioneered bench-scale production of baculovirus- expressed proteins about seven years ago. "Previously, the cells were grown in little spinner flasks," he says. Clemm is now able to perform biological characterizations, assay development, and structural studies with the larger quantities of protein material obtained from his 10-liter system. A steady supply of high-quality, reproducible recombinant proteins is necessary for applications such as drug screenings that require a lot of material, notes Clemm.

Other researchers also emphasize the flexibility of their bioreactor systems in expanding their investigations. "The Ap- plikon equipment has really been useful for us," claims Mike Vandiver, manager of cell-culture process development at ICOS Corp., a therapeutic drug maker in Bothell, Wash. In addition to working with bioreactor vessels ranging from 3 to 100 liters for his Chinese hamster ovary (CHO) cell-culture studies, Vandiver readily converted several large cell-culture tanks to microbial-process systems for certain other experiments.

When gravity is removed or reduced, as in space travel, life systems degrade at a remarkable rate, very much like a rapid aging process or what occurs after severe trauma or infection. Investigators at Walter Reed Army Institute of Research (WRAIR), in a collaborative effort with the National Aeronautics and Space Administration, are studying the effects of space flight on human cells using artificial capillary cell-culture systems prepared by Germantown, Md.-based Cellco Inc.

This hollow-fiber technology allows human cell growth to be studied outside of the body by simulating the natural three- dimensional function of the human capillary system, supplying oxygen and nutrients to the cells, while removing waste products. The cells grow within a network of artificial blood vessels that are perfused with an oxygen-rich nutrient fluid.

Dubbed the Space Tissue Loss project (STL), the program studies the pathological changes that develop in various cell types at zero gravity. It could also reveal how space flight can cause the tremendous loss of calcium and minerals from bones, and find ways to prevent or minimize bone failure in space and on Earth. Results from tests of muscle deterioration could yield more information about similar muscle failure that occurs in muscular dystrophy, the loss of muscle mass after severe injury, prolonged bed rest, and aging.

"We realized quite early there was no hardware available to do these experiments on the space shuttle, so we had to develop a system that would actually perform a controlled experiment on board," says Col. William Wiesmann, STL program director and head of WRAIR's surgery division. While devising the self- contained cell-culture apparatus, the researchers arranged with NASA to fly some investigators, using the equipment to study the effects of microgravity on a variety of tissue types, including human donor stem cells and muscle cells for primary mature cell cultures.

Scientists from the Naval Medical Research Institute (NMRI) are also investigating the growth and development of bone marrow stem cells with experiments aboard shuttle flights. "Astronauts exposed to microgravity develop persistent hematologic abnormalities. For example, they become anemic and their lymphocytes don't function normally," observes Kelvin Lee, head of NMRI's Stem Cell Biology Branch.

The goal of NMRI's current study is to examine how microgravity affects he-matopoiesis the generation of the cellular components of blood occurring within bone marrow. Naval researchers and Cellco scientists developed an in vitro hematopoietic microenvironment culture system that mimics the bone marrow microenvironment. NMRI scientists are focusing on the performance of the culture system, in which human bone marrow cells are cultured on top of a "feeder" monolayer of microvascular lining cells. An Army and Navy collaboration, the NMRI system is being used in conjunction with WRAIR's STL hardware.

"For military deployments, the bone marrow culture system is a perfect device that can be put in forward areas, because it is small and self-contained," Lee notes. "If it will work in orbit, it will probably work on a hospital ship."

Results from this space launch study could aid in the development of new therapies. From a small sample of a patient's own blood marrow, the culture system has the potential to rapidly grow quantities that can be transplanted back into the patient. This type of treatment, called ex vivo hematopoietic cell expansion with autologous bone marrow transplantation, could be used to treat battlefield casualties with much fewer complications than occur with current treatment.


The CelliGen Plus Bioreactor, a commercial-scale system from New Brunswick Scientific Co. of Edison, N.J., uses several cell-growth technologies to produce high cell densities with numerous cell lines. When the unit is used as a standard stirred-tank system, typical yields of 107 cells per ml have been reported from a variety of mammalian, insect, and plant cell lines. With the addition of a packed-bed basket, the system can be used as an immobilized-cell bioreactor, reducing shear damage to cells and producing up to 1 gram per day of secreted products, the company reports.

"We were able to achieve viable cell levels of between 5 and 12 x 107 cells per ml inside the basket for a number of different cell lines, including hybridomas and recombinant CHO lines," says Richard Kirschner, a senior research scientist at Kalamazoo, Mich.-based Upjohn Co. "That's certainly a high density as compared to other approaches, and much more than we could get from a stirred-tank situation." Citing one study in particular, Kirschner "carried out the experiment for 42 days, and the cell number was 1011 total, in a 1.75-liter basket."

For research needs, New Brunswick Scientific offers a spinner basket that produces milligram quantities of secreted products. Like the CelliGen Plus, this screening tool utilizes immobilized cell technology, trapping cells on a bed of polyester disks.

Susan Keznoff, an account manager at Minneapolis-based Cellex Biosciences Inc., reports that the company recently introduced a universal bioreactor cell-culture system with the flexibility to utilize four different cell-culture technologies, including hollow fiber: "This fully automated control system is suitable for the experimental selection of the best bioreactor technology for a given cell line, as well as research to pilot levels of production."

Because cells have a natural tendency to grow in three dimensions, the inherent limitations of two-dimensional substrates--such as T-flasks, petri dishes, and multi-well plates--tend to curtail the yields of layered cultures. As cells pile on top of each other, those in the lower layers often perish quickly because of the slow diffusion of oxygen and nutrients.

To overcome these deficiencies, manufacturers now offer a variety of artificial capillary cell-culture systems. These hollow fibers substantially facilitate in vitro cell growth by maintaining a stable microenvironment around the cells. "Researchers are switching from static plates, roller bottles, and mice to this technique," says Alberto Correia, a vice president at Unisyn Technologies in Milford, Mass. Previously, the technology was not readily scalable to the required levels, and scientists were hesitant to use it until they were certain the process could be transferred into production, he points out.

Apparently, that hurdle has now been successfully conquered. For example, scientists at Roche Diagnostics in Branchberg, N.J., use the Unisyn system to conduct large-scale production activities. They maintain it's a cost-efficient means of growing nearly pure antibody, with an almost 70 percent savings over ascites mice.

And at Goodwin Biotechnology, a contract manufacturing company located in Plantation, Fla., vice president David Fischer explains that "as we bring a client's cell line in-house, we produce [the cells] primarily in hollow-fiber bioreactors." The system is easier and less expensive than a stirred tank system, notes Fischer, and the volume of material produced is substantially more than most organizations require.

Stuart Rosenfeld, a principal research scientist at DuPont Merck Pharmaceutical Co. in Glenolden, Pa., describes the advantages of using hollow-fiber membranes to produce recombinant proteins from cells: "Less labor is required to maintain them; less serum additive is needed for the medium; and the secreted product is in a more concentrated form, making purification much easier."

"In general, the advantages of this technology are very much underutilized," maintains Mark Keck, the director of marketing for Cellco Inc. in Germantown, Md. "It's simply a better way to grow cells, and that could be any cell." Researchers will soon discover this technology really has some broad applications, Keck maintains. For example, "we're already seeing some excellent results in the field of endothelial cell study."

"We use the Cellco equipment to culture endothelial cells on the inner surface of the hollow fibers," says Barbara Ballerman, an associate professor of medicine at Johns Hopkins University. "This process allows me to impose the same physical forces of shear stress and pressure on the cells as would normally occur in vivo, because the medium flows past the cells just as blood would flow past them as they line the blood vessels."

Using this technique, Ballerman now can study the endothelium and its mechanism of attachment to the surface on which the cells are grown. "The attachment is being stimulated by the physical forces, so I have a model system to study that process," she notes.

Rick Shuman, a research scientist in charge of monoclonal antibody production at Virion Systems Inc. in Rockville, Md., sums up the advantages of hollow-fiber techniques this way: "We're growing cells in a defined medium. They're sterile when they go in and sterile when they come out, and it's much simpler to purify the secreted antibodies."

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