Cell And Tissue Culture Techniques A Combination Of Science And Art

Science And Art Author: Holly Ahern Cells of all types -- from organisms as simple as bacteria to those as complex as humans -- can be removed from representative tissues and grown in a culture vessel, where they reproduce and perform the same biological functions as cells in their natural state. When human skin cells such as fibroblasts are grown in culture, for example, they attach to the culture vessel and form a layer, just as if they were forming a layer of skin. Cultured fibroblasts secrete proteins such as fibronectin and fibroblast growth factor in the same way that cells in tissues do. This property of producing proteins in vitro makes cultured cells an attractive biopharmaceutical tool. The cells act as tiny protein factories that churn out liters of valuable antibiotics, hormones, growth factors, and other chemotherapeutic agents. Growing cells in culture is an exacting science that requires, in addition to skill in aseptic technique, special glass or plastic culture vessels; nutrient-rich media to bathe the cells as they grow; and incubators to keep the cells warm (or cold, if the cells prefer). Products such as these, specially designed for cell culture, are commercially available from several biological and biomedical supply companies. Because room air teems with microscopic life forms that can by chance fall into an opened culture vessel, many cell biologists prefer to work with their cultured cells in a hood or sterile cabinet, to keep the airborne contaminants out. HEPA EQUIPPED: Labconco Purifier safety cabinets, available from VWR Scientific, come with high-efficiency particulate air (HEPA) filters. "When a cell culture is contaminated with microbes such as bacteria or fungi, the cultured cells generally die because the microbes use up the available nutrients and secrete toxic metabolites into the culture medium," explains Don Orokos, a cell biologist at the State University of New York, Albany. Consider an experiment performed with cultured cells to study the process of aging. To "age" cells, they must be repeatedly transferred from one culture vessel to a fresh vessel, to replenish nutrients and to provide more space for growth. Now imagine that after months of subculturing to achieve aged cells, a contaminant slips in and ruins the culture. To prevent such occurrences, cell biologists culture their cells in an enclosure such as a hood, which offers a circulation- free, nonventilated environment for culturing cells. "Working in a hood, where the work space can be rendered virtually sterile, drastically decreases the chance that a culture will become contaminated," Orokos notes. Laminar flow hoods, with recirculating air patterns limiting horizontal movement of particles in the hood, are especially useful when the cultures are a potential biohazard, including virus cultures or virus-infected cells, or bacterial pathogens such as Mycobacterium tuberculosis. Ventilated hoods often include HEPA (high-efficiency particulate air) filters, to capture microbes in the air before they can enter the hood and contaminate carefully cultured cells. SMOOTH SURFACE: Labconco's Protecto tissue-culture enclosure is fire- and chemical-resistant and crevice-free for easy cleaning. Hoods utilized for cell- and tissue-culture work are available from Pittsburgh-based Fisher Scientific, which distributes products for Labconco of Kansas City, Mo., including laminar flow and nonventilated tissue-culture hoods. Labconco Protector tissue-culture enclosures are made of fire- and chemical-resistant fiberglass that is smooth and crevice-free for easy cleaning and sterilization. Included is a germicidal ultraviolet light that researchers can switch on at the end of a culturing session to sterilize the interior of the hood. The Protector tissue-culture enclosure is also available through VWR Scientific, based in West Chester, Pa. Labconco's Purifier safety cabinets, available from Fisher and VWR, come with laminar-flow or total-exhaust capabilities, in which the air flow is directed up and away from the cultures in the work space. Both are equipped with HEPA filters that reportedly remove from the air 99.99 percent of the airborne particles larger than 0.3 micron before they can enter the hood. This size range accounts for all bacteria and many types of virus. Bellco Glass Inc. of Vineland, N.J., markets Bellco Biological Safety Cabinets, which provide a localized, particulate-free, sterile environment for tissue-culture work. Bellco's patented HEPAire filtration system, a zero-bypass, leak-proof filter unit, is incorporated into the safety cabinet to ensure that no contaminated air bypasses the HEPA filter and enters the hood (or, alternatively, escapes into the laboratory) to contaminate cultures, according to the company. Bellco will outfit safety cabinets and clear-air systems for customers whose requirements range in magnitude from a single unit in one laboratory to entire buildings. When neurobiologist R.G. Harrison of Johns Hopkins University in Baltimore created the first batch of cultured cells in 1907, he grew nerve cells in culture vessels made of glass and used a medium of clotted lymph fluid, rich in proteins, salts, and water (R.G. Harrison, Journal of Experimental Zoology, 9:787-848, 1910). Five decades later, Harry Eagle of the National Institutes of Health found that animal cells could be propagated in a much more defined mixture of amino acids and salts when his medium was supplemented with serum (H. Eagle, Proceedings of the Society for Experimental Biological Medicine, 89:362, 1955; H. Eagle, Science, 130:432, 1959). The goals of these cell-culture pioneers and those of successive generations of cell biologists were to create an artificial environment similar enough to the cells' natural environment to permit their continued growth and proliferation. The objectives of cell culturists have not changed much over the course of the century, but their choice of tools most certainly has. Today's cell biologists may choose to grow cells in plastic flasks specially treated for tissue-culture applications, on glass or collagen-coated plastic beads, or in hollow-fiber cartridges, through which culture media can flow. Media choices have expanded greatly, and now include formulations that will support the growth of a wide range of cells, from human fibroblasts to plant and insect cells. "The culture media is the cells' source of nutrients," says Orokos. The basic formula of many commercially prepared culture media consists of buffered salts and amino acids, to which supplements such as serum (the liquid portion of blood), antibiotics, or growth factors can be added. Just as people have varying preferences for food, cultured cells have different nutritional requirements for growth. Insect cells, such as those of Spodoptera frugiperda (Sf-9), prefer media rich in amino acids and vitamins but have a minimal requirement for additional serum. Mammalian cells, however, thrive when there is a significant concentration of serum in a medium (5 percent to 20 percent). Serum, which can be obtained from the blood of any mammal, is considered a Pandora's box of sorts when it comes to cell culture. Although serum contains substances such as attachment and growth factors, nutrients, minerals, amino acids, and hormones, which are known to aid cell cultures, some scientists are concerned that adding such an undefined mixture to their cultured cells could lead to unanticipated (and perhaps disastrous) results. "As a supplement, serum provides additional growth factors for cells," explains Anita Vizzini, technical specialist at Life Technologies Inc., a supplier of culture media and reagents in Gaithersburg, Md. "However, serum is not well-defined in a chemical sense, and some researchers prefer to use media that is specially formulated to support the growth of cells without added serum." Life Technologies carries a broad line of buffers, culture media, serum, and supplements. The company's media include formulations for culturing many different types of cells, such as fibroblasts and blood cells from mammals, monoclonal antibody-producing hybridoma cells (created by fusing immortal human cells with antibody-secreting mouse cells), plant cells, and genetically engineered insect cells. Life Technologies will also prepare culture media to the specifications of its customers. "An investigator performing nutritional studies on cells may require a [medium] that lacks a particular amino acid, or might require a large quantity of media to scale up protein production from cultured cells," says Vizzini. "For these customers, we will provide custom formulations or packaging to suit their application." Sigma BioSciences of St. Louis also has a wide range of liquid and powdered media, serum, buffers, growth factors, and other defined-medium supplements available for researchers who want to grow cells. Sigma's Hybri-Max line of reagents consists of products that are specifically formulated for hybridoma production, such as conditioned medium (the supernatant removed from cells already in culture) for increasing hybridoma survival and enhancing their growth. The Hybri-Max line also includes reagents such as the cell-fusing agent polyethylene glycol (PEG) and quabain, a glycoside that is used to select for human/mouse hybridomas after they have formed. Powdered-media and liquid-media formulations for growing all types of cells (mammalian, plant, and insect) are available from ICN Biomedicals of Costa Mesa, Calif., which carries a large selection of cell-culture products. ICN's CELLect Gold fetal bovine serum is guaranteed to be free of mycoplasma (a bacterial contaminant that often invades cell cultures) and virus, with no detectable endotoxin (a bacterial toxin). CELLect sera are also collected from newborn calves and iron- supplemented calves, as well as from horses, chicken, goats, pigs, and humans. ICN also has available media deficient in phosphates or amino acids, such as the Methionine/Cysteine Deficient Medium, Leucine Deficient Medium, and Phosphate Deficient Medium, which are utilized in experiments on cultured cells in which researchers wish to label cellular proteins or DNA with an isotope. For assays, such as the metabolic labeling of mammalian, bacterial, or yeast cells in culture, researchers can combine a Deficient medium with a particular radiochemical label. For example, to label DNA in cultured cells, an investigator might choose Phosphate Deficient Medium to be used in association with ICN's 32P Orthophosphate radiolabel. Many types of cells require a solid surface on which to adhere before they can be grown in culture. This solid support mimics the cells' natural environment in the tissue from which they were derived. Human fibroblasts, for example, require a surface to which they can attach. In the body, they are found in skin layers, where they flatten and spread to form a sheet of sorts. With the exception of blood cells and some types of transformed (cancerous) cells, cells in culture need a solid support. Currently, plastic is the support material of choice for scientists performing cell culture. The flat surfaces of tissue-culture flasks, trays, petri dishes, multiwell culture plates, and even the inside surfaces of large roller bottles, make ideal support surfaces for growing cells. Cells will also to grow on the surfaces of microcarrier beads made of collagen, glass and plastic, and in the lumen of hollow-fiber growth cartridges. These latter surfaces are generally used by researchers who grow cells to high density, to collect their protein products. RIBBED SURFACE: Corning Costar's ExCell flasks are said to increase a cell's growth surface. Several types of tissue-culture vessels are available from Fisher. The company distributes an extensive selection of plastic culture dishes, plates, flasks, and roller bottles for Cambridge, Mass.-based Corning Costar Corp.; the Falcon line of tissue-culture ware from Becton Dickinson Ltd. of Oxnard, Calif.; and plastic products from NUNC Inc. of Naperville, Ill. Corning's new ExCell flasks are designed with a ribbed surface that is claimed to increase the cells' growth surface by 75 percent over conventional tissue-culture flasks. Although cells readily attach to most plastic surfaces, many cell biologists prefer to use products with specially treated surfaces, which promote more secure attachment of cells. Treated and untreated supports can be purchased from Fisher, VWR, and the individual suppliers. VWR also distributes Corning and NUNC tissue-culture products, such as NUNC's Cell Factories, which are a stack of culture trays that share a common inlet and outlet port. The Cell Factories provide cells with a growing surface as large as the inside of a roller bottle, but take up less space in the incubator. Also available from VWR are larger-scale Nalgene culture vessels (from Nalge Co. in Rochester, N.Y.) and products for large-scale cultures from Wheaton Science Products of Philadelphia, in which the medium surrounding cells growing on flask surfaces or on microcarrier beads are gently "stirred," to optimize the growth conditions. The Nalgene Culture Vessel with Biotech Mixer is a complete mixing system for a 12-liter culture of cells. Wheaton's Biostir and Microstir Stirrers are units that can accommodate up to four flasks (such as Wheaton's Celstir double-sidearm flasks) and are designed to conserve incubator space. For growing cells in a culture plate or in wells on a microplate, lining the surface first with a clear membrane makes manipulating the cells much less difficult. Once the cells have attached to the membrane's surface, they can be removed for visual evaluation of cell growth or to stain the cells with biological or chemical dyes. Becton Dickinson's Falcon line's Cell Culture Inserts are membrane culture-plate inserts that are available in three pore sizes, to allow maximum permeability of nutrients both large and small. The Anocell Porous Tissue Culture Insert from Whatman Inc. of Clifton, N.J., is a rigid, uniform surface for cell growth that can easily be removed from the culture vessel for experiments. Both of these products are also available through VWR. In most cases, the goal of the researcher growing cells in culture involves much more than simply removing cells from a tissue and keeping them alive for extended periods. Cell culture has been considered an alternative to product testing in animals, because it is logical to assume that cells grown in an artificial environment are models of the tissue from which they are derived. However, because the cells in culture are removed from their natural state, the downside is that it would be illogical to assert that cultured cells and cells in tissues behave in identical ways. Measuring the death of cells in culture, however, can be used as an experimental indication of a substance's toxicity. With the CytoTox 96 Non-Radioactive Cytotoxicity Assay from Promega Corp. of Madison, Wis., investigators can measure the release of the enzyme lactate dehydrogenase (LDH) from cultured cells as a sensitive and accurate marker of chemical toxicity. Using the CytoTox 96 kit, researchers can assess the effects of a particular substance, perhaps a potential biopharmaceutical, on mammalian cells exposed to varying concentrations of test material over a period of days. The release of LDH into the culture supernatant correlates with the amount of cell-membrane damage and cell death. "This provides an accurate measurement of the cellular toxicity of the test substance," according to Kimberly Huston, a technical-services scientist at Promega. One area in which cells in culture excel is in the production of biopharmaceuticals and research biochemicals, such as enzymes, hormones, and antibodies. Such proteins might be a natural product of a particular cell, or secreted by genetically engineered bacterial and insect cells that can express human proteins from a cloned gene. Proteins produced by genetically engineered cells include chemotherapeutics -- such as insulin and erythropoietin used by physicians to treat human disease -- or highly specific monoclonal antibodies that will detect a single epitope on an immunogenic substance. Because the proteins secreted by cultured cells are usually present in low quantities, many researchers clone genes encoding valuable proteins into an expression system, such as a bacterial plasmid or a virus, where the gene will be overexpressed. One such system is the baculovirus expression system, in which genes are cloned into a virus that infects insect cells. Insect cells carrying the cloned gene grow in culture and secrete the protein of interest, which can be collected from the culture supernatant and purified for use as a biopharmaceutical or biotech product. The Bac-to-Bac System from Life Technologies is based on a "bacmid" expression system, in which a target gene is first cloned into a donor plasmid (pFastBac1) and then transformed into E. coli cells. Once inside the cells, the gene is transferred from the donor plasmid to the baculovirus shuttle vector (the bacmid) by site-specific transposition. The recombinant bacmid DNA is isolated from the cells and inserted (transfected) into insect cells. When harvested from the transfected cells, the recombinant baculovirus is used as a stock culture for the subsequent infection of insect cells. The infected cells produce the desired protein and secrete it into the culture supernatant, from which the protein can be collected and purified. "To produce proteins, there are several advantages to using a baculovirus expression system with cultured insect cells," says Brian Schmidt, technical specialist in molecular biology at Life Technologies. Baculoviruses have a restricted host range and are safer to work with than other types of mammalian viruses. When expressed in insect cells, the protein product of the cloned gene is chemically modified as it would be in vivo. In addition, insect cells are easy to maintain in culture (they can be grown at room temperature and without CO2) and therefore cost less to grow than mammalian cells. Concludes Schmidt: "Overall, it's just a nice cell system." For some life scientists, growing cells in culture is a way to obtain the true object of their research -- a product of a cloned gene. For others who study the activities of cultured cells to learn more about how cells regulate their growth and development, and how they communicate in vivo, cell culture is a combination of science and art. Holly Ahern is a science writer and an assistant professor of biology at Adirondack Community College in Queensbury, N.Y.

Cell And Tissue Culture Techniques A Combination Of Science And Art

Interested in reading more?

Become a Member of