One of the most important advances in the field of cell biology came in the early 20th century, with the discovery that plant and animal cells could survive - and even replicate - outside the living organism. In 1907, R.G. Harrison, a neurobiologist trying to prove that nerve fibers were actually outgrowths of single cells, chopped up spinal cord tissue and added it to clotted plasma in a humidified growth chamber. The nerve cells from this crude explant not only grew and divided in this environment, but also extended long axons into the plasma clot in a manner comparable to that seen in vivo, clearly showing for the first time that cells could be grown in an artificial environment. Glass or plastic culture vessels eventually replaced plasma clots, and specialized growth media - containing a complex balance of amino acids, vitamins, salts, and minerals - were developed to support the growth of cells in vitro.
With the advent of genetic engineering and hybridoma technology in the late 1970s, cell culture has taken on a whole new meaning. Genetically engineered mammalian cells produce and secrete proteins of therapeutic importance; proteins such as interferon, growth hormones, erythropoietin, and some types of vaccines. Hybridoma technology, the science of fusing immune mouse cells to immortal cancer cells, made it possible to produce monoclonal antibodies from cells grown in culture. To produce these proteins in the quantities required for commercial use, it became necessary to scale up cell culture efforts. The search for a simple, cost-effective cell culture system has resulted in the birth of a whole new area of biotechnology: the use of bioreactor systems to grow cells on a large scale.
An important recent advance in large-scale cell culture came with the advent of hollow fiber-based growth systems. Hollow fiber technology mimics the capillary-type circulatory systems found in vivo. Hundreds or thousands of hair-like hollow fibers, which are permeable membranes made of substances such as cellulose acetate or polypropylene, are bundled together into glass or plastic cylinders to form a cartridge. The cartridge is then attached to a perfusion system, which circulates nutrients vital to cell growth through the fibers in a continuous flow of media.
Cells are inoculated on the outside of the fibers in the cartridge. Nutrients such as glucose and amino acids, which have a low molecular weight, diffuse across the membrane of each fiber and nourish the cells. At the same time, metabolic waste products are transported across the membrane in the opposite direction into the circulating media, and are carried away from the cells. Because of the size exclusion capabilities of the permeable membrane, desired protein products with a high molecular weight, such as antibodies, can be excluded from passing into the lumen of the fibers, and thus become concentrated in the extracapillary space. The concentrated product can then be collected and further purified in large quantities.
All of the hollow fiber systems on the market are based on the same general principle: to provide cells with a system that simulates the tissue from which they came. The system keeps them oxygenated and supplied with essential nutrients, while removing waste products via perfusion. At the heart of such systems is the hollow fiber bioreactor, the cartridge in which the cells actually grow. Three manufacturers - Kinetek Systems of St. Louis; Amicon of Beverly, Mass.; and Endotronics Inc. of Minneapolis - all offer perfusion-based, integrated cell culture systems. These systems incorporate the hollow fiber bioreactor, pumps to circulate media, incubators, media reservoirs, and gas exchange components into an integrated, microprocessor-controlled benchtop package. Built-in quality control features and options, such as pH and temperature monitoring, can be added to fully automate the cell culture process. While all use similar technology, each system has features that differentiate it from the others.
Kinetek Systems manufactures two families of hollow fiber bioreactors to accompany its cell culture system, the Network 2000. The Hybrinet bioreactor supports high-density growth of suspension and anchorage-dependent mammalian, plant, or insect cells. The Anchornet bioreactors are patented hollow fiber membranes designed specifically to support the growth of anchorage-dependent cells, which are often hard to grow in bioreactors because of their solid surface requirement. The Anchornet membranes carry a charge (positive, negative, or neutral) to optimize cell attachment and growth conditions for particular sets of cells. The base price of the Network 2000 system is $8,300 (installed). Available optional features, including a high-efficiency air particle filter, a "feed and bleed" module (to pump media through the system), and a pH and perfusion module (to monitor and control pH), can tack on an additional $6,000 to $10,000, yet make the system virtually fool-proof. A similar - but smaller-scale - system recommended for mono- clonal antibody production, the Network 1000, is also available at a cost of $1,975.
The Vitafiber VLS system, from W.R. Grace and Co.'s Amicon Division, is based on a "Flo-Path" bioreactor, a hollow fiber cartridge hooked up to a preassembled unit that includes an oxygenator, tubing, and valves. The Flo-Path bioreactor, available from Amicon in three sizes for various applications, can be purchased for $900 to $1,300 (excluding pumps), and represents an economical alternative to higher-priced, fully automated systems. The Flo-Path bioreactor, when attached to the Vitafiber VLS system, becomes a computer-controlled integrated system, available for $20,000. Addition of an optional pH probe ($450) makes the system fully automated.
Endotronics Inc. markets three hollow fiber systems: the Accusyst P/3X, for production-scale application ($125,000); the Accusyst-Jr., for pilot-scale applications ($30,000); and the Accusyst-R, a bench-top system for research-scale applications. The Accusyst-R, available for $14,000, is comparable to the Vitafiber and Network 2000 bench-top systems. According to Endotronics, one to five grams of protein product can be produced from this system per month. Three different sizes provide scientists with a full range of cell culture capabilities, to better meet the needs of individual laboratories.
Hollow fiber technology represents a major breakthrough in the science of cell culture, especially in the production of monoclonal antibodies. Studies have shown that hybridoma cells grow to very high densities in hollow fiber bioreactors, and produce large quantities of a specific antibody, which can be collected easily and further purified if necessary. Use of serum-free media can eliminate the purification step for many applications, which translates into real savings in terms of time and overall cost of monoclonal antibody production.
In addition, hollow fiber systems have provided scientists with a means of studying the complex behavior of cells in a strictly controlled environment, providing new insights into cellular physiology. Though still in its infancy, this technology should provide the groundwork for even greater advances in the study of cells and their protein products.
Holly Ahern teaches immunology and cell and molecular biology at the State University of New York, Albany.