Push For Genetically Engineered Therapeutics Drives Cell-Culture Market

Cell-Culture Market Biotechnology and biopharmaceutical companies are putting the pressure on manufacturers of cell-culture equipment and supplies to create products that will enable researchers to culture cells more efficiently and in large amounts. The increased need for these products is an outgrowth of the growing demand for genetically engineered products with proven or potential therapeutic value. The laboratory products industry is responding with new types of cell-culture vessels that boast specially treated surfaces to which cultured cells may firmly adhere. Also on the market are new formulations of culture media, as well as instruments that allow researchers to grow vast quantities of cells while continuously collecting the prized protein product that the cells secrete as they grow in culture. Several types of cells, from sources as diverse as bacteria and humans, retain their ability to synthesize and secrete the protein products characteristic of the tissue from whence they came. In many cases, the proteins that cultured cells secrete are specially created by recombining genes from different organisms. For instance, the insulin product used most often to treat human diabetics is mass-produced by bacteria that have been genetically engineered to carry and express the human genes required for the synthesis of this substance. Monoclonal antibodies, useful as research tools and as biopharmaceutical agents, are secreted in culture by cells called hybridomas. Hy-bridomas are created by fusion of antibody-producing lymphocytes with immortal mouse cancer cells. In the area of basic research, cell culture has become a more exact science than ever before. "For many years, getting cells to grow in culture was more of an art than a science, performed with culture media containing serum, which is largely undefined in a chemical sense, and requiring technical skill and patience on the part of the investigator to determine the best set of artificial growth conditions for a particular type of cell," comments cell biologist Don Orokos, a laboratory director in the department of biological sciences at the State University of New York, Albany. "However, with the development of new formulations of culture media for which the exact chemical composition is known, along with the advanced plastic cultureware currently available and new techniques for keeping cells growing, the practice of cell culture has become a science." A wide variety of culture media in formulations to support the growth of several different types of cells can be purchased from companies such as Life Technologies Inc. of Gaithersburg, Md., which produces the Gibco/BRL line of cell culture media and products. Other vendors of culture media are ICN Pharmaceuticals Inc. of Costa Mesa, Calif., and St. Louis-based Sigma BioSciences, a company with a broad product range that includes several types of liquid and powdered culture media, serum, and several defined medium supplements, such as amino acids and vitamins. USER-FRIENDLY: The EZ Access Reactor from Bellco Glass reportedly can be set up in half hour Researchers can obtain plastic and glass cultureware suitable for growing all types of cells from glassware and plasticware manufacturers such as Bellco Glass Inc. of Vineland, N.J., and Corning Costar Corp. in Cambridge, Mass., as well as from larger laboratory product distributors like Pittsburgh-based Fisher Scientific Inc., VWR Scientific of West Chester, Pa., and Krackler Scientific Inc., headquartered in Albany, N.Y. Although the so-called basal media-which are pH-balanced mixtures of salts, amino acids, sugars, and vitamins-provide cells with many of their nutritional requirements, they do not support cell growth over a long term. To these media, serum (the liquid portion of blood) must also be added. Serum is known to contain a wide variety of biological substances, such as proteins, salts, attachment factors, and hormones, along with several hundred other "growth factors," which apparently help cells, particularly those of mammalian origin, to survive outside of their representative tissues. When cell-culture media are supplemented with serum (in the range of 5 percent to 20 percent), a significant proportion of the medium becomes chemically undefined. To limit the potential for unexpected results, such as the introduction of inhibitory factors (as mysterious as the growth factors) in a cell-culture experiment, some researchers prefer to use media for which the exact chemical composition is known, called serum-free media. "Serum-free media can be defined as a complete formulation capable of supporting the growth of cells in culture without the addition of serum supplements," explains Anita Vizzini, technical specialist at Life Technologies. Serum-free media formulations provide researchers with several advantages over conventional culture media to which serum must be added. With a serum-free medium, cell culture is a defined process that provides for greater consistency between experiments. Using media without serum also makes it easier for researchers to purify a desired protein product from a culture after the cells secrete it into the supernatant. Life Technologies provides several different formulations of serum-free media. AIM V Medium is a serum-free formula that was designed specifically for culturing lymphocytes (a type of immune cell) in clinical trials of adoptive immunotherapy, a potential method for treating cancer. AIM V Medium has also been used successfully for the serum-free growth of many other types of cells, including mouth epithelial cells and human fibroblasts (skin cells), and immune cells, like macrophage and hybridoma cells. To grow nervous system cells, Life Technologies' Neurobasal Medium is a serum-free basal medium that is specially formulated to support the growth of hippocampal neurons and other types of neurons. Life Technologies offers additional formulations for investigators who wish to grow keratinocytes (skin cells), hepatocytes (liver cells), and vascular endothelial cells. The company will also prepare custom formulations of serum-free media. "An investigator performing nutritional studies on cells may require a serum-free culture medium that lacks a certain amino acid," says Vizzini. "For these customers, Life Technologies will provide custom formulations or packaging to suit their application." SELECTED SUPPLIERS OF APPARATUS, MEDIA, AND RELATED SERVICES FOR CELL AND TISSUE CULTURE American Fluoroseal Corp. American Type Culture Collection Becton-Dickinson Ltd. Bellco Glass Inc. Celox Laboratories Inc. Corning Costar Corp. Fisher Scientific Inc. ICN Pharmaceuticals Inc. IGEN Inc. Krackler Scientific Inc. Life Technologies Inc. NUNC Inc. Pharmacia Biotech Inc. Sigma BioSciences Synthecon Inc. Vistra Biologicals Corp. VWR Scientific ICN Pharmaceuticals offers serum-free media and serum extenders for laboratory-scale cell culture as well as researchers interested in scale-up applications. ICN's BioRich 1 medium is enriched with trace elements to help cells adjust to serum-free conditions. According to ICN, BioRich 1 supports cell growth comparable to that achieved in standard culture media containing 10 percent serum. BioRich 1 can be supplemented with ICN's ITS Premix (which contains the essential growth factors insulin, transferrin, and selenium). With the supplement, BioRich 1 becomes a complete medium that will support the growth of many types of cells in a serum-free environment. One of the problems associated with serum-free cell culture is the response of the cells when they are removed from serum-bathed tissues or subcultured from cells previously grown in the presence of serum. Many types of cells will not tolerate an abrupt transition to a life without serum, and so the cells must be slowly weaned into culture medium containing gradually decreasing amounts of the substance. For investigators who utilize some of the more common cell lines, such as HeLa (an immortal cell line derived from a cervical tumor), the American Type Culture Collection (ATCC) of Rockville, Md., and Celox Laboratories Inc. of Hopkins, Minn., have teamed up to produce cell lines that are pre-adapted to life without serum. ATCC Serum-free Cells are available in kits that also contain Celox's serum-free growth medium and serum-replacement reagent. Serum-free Cells are available in three cell lines: ATCC cell line CCL-81.1K, commonly known as Vero, originally derived from African green monkey kidney cells; ATCC's HeLa cell line CCL-2.3K, derived from a human cervical epitheloid carcinoma; and an MDCK cell line, ATCC CCL-34.2K, derived from canine kidney cells. One area in which serum-free media have had an impact is in the mass production of monoclonal antibodies. Antibodies, which are produced naturally by a particular subset of immune cells called B cells, are powerful reagents in research applications and in biomedicine because of their exquisite specificity for a single target. Monoclonal antibodies enhance this power by binding to only one site among the many available on the surface of the target molecule or cell (the antigen). Monoclonal antibodies are synthesized by cells called hybridomas that are created by fusing antibody-secreting B cells with cancer cells that divide indefinitely. After the cells are fused, the resulting hybridomas are screened for the presence of the desired antibody. A selected hybridoma "clone" can be expanded in cell culture into millions of cellular antibody factories that churn out liters of the monoclonal antibody product. Since the hybridomas secrete their antibodies directly into the culture medium, the advantages of using a serum-free formulation are immediately apparent. "Many of the expression systems used most commonly in the production of recombinant proteins are CHO [Chinese hamster ovary] cells or insect cells, both of which can be grown in serum-free and protein-free media," comments Robert J. Fleischaker, president of Carlsbad, Calif.-based Vistra Biologicals Corp. Vistra is a company that provides contract cell-culture services, including monoclonal antibody production. Vistra's customers include small genetic engineering firms as well as large biopharmaceutical companies. These companies contract with Vistra because they lack cell-culture or protein-purification expertise, or to keep up with unexpected or growing demand for their products. "The use of serum-free media significantly simplifies the process of purifying the product from the culture supernatant," says Fleischaker. "From a regulatory point of view, using a defined medium makes it easier to win [Food and Drug Administration] approval for a biological substance because the components of the raw materials were well defined." Additionally, to produce large amounts of a particular monoclonal antibody or other biopharmaceutical would require huge volumes of serum, more than could be obtained from a single animal. Thus, batch-to-batch variations in the added serum could lead to inconsistent production of the antibody by the cultured hybridomas. "There is also the slight risk of transmitting animal viruses or infectious particles called prions to humans when animal sera are in the production process," Fleischaker adds. FINDING A HAPPY MEDIUM: Sigma BioSciences offers a selection of culture media. Sigma BioSciences carries several serum-free and protein-free media specifically created for the culture of hybridoma cells. QBSF 52 is a low-protein, serum-free medium designed to increase the production of monoclonal antibodies from hybridomas created from fusions involving cells in the mouse myeloma cell line NS-1. Similarly, QBSF 55 medium from Sigma was formulated for hybridomas made from mouse cells in the SP 2/0 line. Sigma's Hybri-Max line of hybridoma media consists of serum-free and protein-free media available in powdered form or as a liquid. They are chemically defined to support several types of hybridoma cells in culture. Hybri-Max products also include hybridoma- enhancing supplements, such as conditioned medium (media removed from cells growing in culture) and B and T Cell Growth Supplement, derived from a mouse lymphoma cell line. Growth enhancers for hybridomas are also available from IGEN Inc. in Gaithersburg, Md., a company that supplies cell culture reagents and products. IGEN's Origen HCF is a hybridoma cloning factor obtained from a conditioned medium in which mouse macrophages were grown. IGEN also produces Origen B&T CGS (B and T cell growth supplement) and CGT-CM (Giant Cell Tumor-Conditioned Medium), all of which are used by researchers to enhance the production and growth of monoclonal antibody-secreting hybridomas in culture. A very important aspect of cell culture is to keep unwanted organisms, such as bacteria and viruses, away from the culture media while allowing circulation of atmospheric gases around the cells. Several companies market plastic labware that provides cells with a sterile environment and the ability to exchange gases such as O2 and CO2. A solid surface to which cells can adhere is also important, because with the exception of blood cells and some types of cancer-forming cells, most cells must be anchored before they will behave as they do in their natural tissues. Fisher Scientific and VWR Scientific distribute an extensive selection of plastic culture dishes, plates, flasks, and roller bottles for Corning Costar Corp. and for NUNC Inc. of Naperville, Ill. Krackler Scientific also distributes tissue culture plasticware by Corning Costar and NUNC, in addition to products in the Falcon line of tissue cultureware, manufactured by Becton-Dickinson Ltd. of Oxnard, Calif. American Fluoroseal Corp. of Columbia, Md., has introduced a cell culture "bag" as an alternative to plastic flasks and bottles. With the Fluoropolymer Cell Culture Bag, researchers can culture cells that lack the attachment requirement, as well as adherent cell lines that can be grown on the surfaces of small beads called microcarriers. The bag is permeable to O2 and CO2, yet its low permeability to water vapor prevents evaporation and allows cultures to grow in air, as opposed to a controlled CO2 environment. Cultures that become contaminated with microscopic invaders such as bacteria, fungi, and viruses lose their value to the investigator because the cells in the culture can never be recovered. Imagine the impact this would have if early on in the hybridoma expansion process, a bacterial contaminant gained access to a culture containing the only hybridoma clone secreting a desired monoclonal antibody. The researcher would have to begin again, from scratch. Mycoplasmas, which are bacteria that lack a cell wall, are common contaminants in cell culture. Because of their cell-wall-less morphology,mycoplasmas have the ability to squirm through membranes and other barriers placed in their way, and often gain access to culture media. Unlike bacteria and fungi, which grow to high cell densities and make their presence known in the culture medium, mycoplasmas usually do not cause turbidity of the culture media or any other of the telltale signs of contamination. However, mycoplasma can insidiously alter a cell's metabolism or growth. Testing for mycoplasma is therefore a necessary step in cell-culture quality control. The Mycoplasma Detection Kit from ATCC is a polymerase chain reaction-based system that detects eight commonly encountered strains of mycoplasma, present in a culture at concentrations as low as 20 colony-forming units (meaning viable cells) per milliliter. Sigma BioSciences offers products for the detection of mycoplasma contamination, including Mycoplasma Agar and Broth, supplied in powdered form, and Mycoplasma Test Broth and Test Medium, which are prepared forms of Sigma's mycoplasma media. ATCC also markets Culture-Check kits for testing cell culture reagents and the cultures for bacterial and fungal contaminants. Some cell lines naturally produce a considerable amount of cellular debris, which might appear similar to contamination. The Culture-Check kit allows researchers to differentiate between cloudiness due to normal cell debris and that which results when bacteria and fungi infiltrate the culture environment. For the production of biopharmaceuticals, cell culture has to be scaled up in order to supply sufficient quantities of cells or cell culture supernatants for processing. This can be accomplished by growing cells on microcarrier beads, which increase the available growing surface, and in large bioreactors in which cells grow and secrete their valuable proteins. Microcarrier particles and macrocarrier beads, in which cells are encapsulated in a polysaccharide coating, provide cells with a large surface area for growth and permit the large-scale cultivation of surface-dependent cells in a relatively small volume of culture medium. In general, higher cell densities in the culture translate into greater yield of the desired product. Cytodex, produced by Pharmacia Biotech Inc. of Piscataway, N.J., is a dextran-based microcarrier particle available in three types. Cytodex 1 is a general-purpose microcarrier that will support the growth of most established cell lines, and is suitable for applications in which the investigator's goals do not include recovering cell products. For research applications in which maximum recovery of cellular products is desired, Cytodex 2 is Pharmacia's recommended microcarrier particle. Cytodex 3 is a microcarrier formulated to support the growth of differentiated or sensitive cells, such as hepatocytes or endocrine cells, that are normally difficult to grow in culture. According to Pharmacia, Cytodex 3 should be the microcarrier of choice when the goal is to recover viable cells from the culture. The Bellco Bioreactor from Bellco Glass is a modular bioreactor system available in several configurations for growing liters of cultured cells. With this bioreactor, cells can be grown immobilized in alginate "Macrocarrier" beads, on a stainless-steel matrix for mildly adherent cell lines, or in a glass cylinder matrix for cells that grow in suspension. One of the options available with this instrument provides researchers with the ability to produce cell-containing alginate beads right in the bioreactor. Cells immobilized in the macrocarrier beads are produced aseptically in the Bellco Bioreactor, and they can be harvested by introduction of a calcium chelator, which dissolves the gel Macrocarrier beads. Also available from Bellco is the EZ Access Bioreactor, a design based on another of Bellco's cell-culture products, the spinner flask. Devices such as Bellco's 1964 and 1965 Spinner Flask models consist of a large glass vessel fitted with internal paddles driven by an overhead motor. The paddles gently stir the cells and cell-culture medium, ensuring optimal circulation of nutrients and essential gases. Spinner flasks can be used to grow both suspension and microcarrier cultures. "The EZ Access Bioreactor is in essence a glorified spinner flask, with a wide flange bearing 18 ports to allow investigators easier access to the culture, in place of the standard 'side arm' design," comments Yelena Fayvilevich, technical support coordinator at Bellco. This bioreactor features a modular control unit, which Bellco considers more "user-friendly" than that of the continuous-flow Bellco Bioreactor. "This system can be set up in half an hour and is quite simple to operate," Fayvilevich explains. "The control modules, which allow the user to set and vary culture parameters such as pH, can be added according to the application, which makes the EZ Access Bioreactor a relatively low-cost design." Houston-based Synthecon Inc. has introduced the Rotary Cell Culture System (RCCS), which is based on a design by scientists at the National Aeronautics and Space Administration's Johnson Space Center, also in Houston. The RCCS bioreactor was developed to maintain cell culture viability before the cells were sent into space on Spacelab or shuttle missions, where cells grow in "microgravity." "Microgravity can be described as free-fall, in a rotary fashion," explains Roger Akers, marketing development manager for Synthecon. "The RCCS provides a 'weightless' effect for the cells. Under these conditions, there are low shear forces, and cells are better able to do what is expected of them, rather than expending energy repairing themselves." While several different cell types have been successfully grown in the bioreactor, "the greatest advantage that the RCCS offers is the ability to introduce cocultures of cells, which then grow into tissues," says Akers. Human liver cells, for example, have grown in the RCCS aggregate into masses of liver tissue up to 1 centimeter in diameter. In addition, the liver tissues that assemble in the bioreactor have the ability to metabolize substances and produce the waste product urea, indicating that the tissue is differentiated and functioning as would normal liver tissue. "The RCCS produces differentiated, three-dimensional cultures of cells that more closely resemble parent tissue," states Akers. He adds that the RCCS is the only system currently available that allows researchers to introduce cocultures that grow into functional tissues. With the RCCS bioreactor, it may be possible to regenerate skin for burn victims, study cancers as they advance from single cells to tumors, and create cell and tissue models for biomedical research applications. New technology that enables the engineering of whole tissues from cultured cells may be the pinnacle of cell culture. It might one day be possible to remove cells from a patient and regenerate functional liver tissue in a bioreactor. Another future possibility is creating artificial cartilage on a disintegrating scaffolding, which could be transplanted into patients whose own tissues have been ruined by disease or injury. "The potential applications of this technology are limited only by the imagination," states Akers.o Holly Ahern is a science writer and an assistant professor of biology at Adirondack Community College in Queensbury, N.Y. (The Scientist, Vol:10, #7, p.18,20 , April 1, 1996) (Copyright © The Scientist, Inc.) WE WELCOME YOUR OPINION. IF YOU WOULD LIKE TO COMMENT ON THIS STORY, PLEASE WRITE TO US AT EITHER ONE OF THE FOLLOWING ADDRESSES: 71764.2561@compuserve.com or The Scientist, 3600 Market Street, Suite 450, Philadelphia, PA 19104, U.S.A.

Push For Genetically Engineered Therapeutics Drives Cell-Culture Market

Interested in reading more?

Become a Member of