Special Report: Cell Biologists Combine Old And New Tools

In 1663, English physicist Robert Hooke viewed cork under a microscope and observed that "these pores, or cells, were not very deep, but consisted of a great many little boxes." Although Hooke's discovery, which involved only the outer boundaries of cells, is considered the beginning of cell biology, it was nearly 200 years before two Germans, botanist Matthias Jakob Schleiden and physiologist Theodor Schwann, focused attention on the microcosm of life in the cell. Hooke, Schleiden, Schwann, and the many others who painted the early portraits of cells would be astounded if they could see the state of cell biology today. Researchers now order cells from catalogs, charging them to grant numbers or credit cards. Cancer cells and blood cells are routinely fused to create a totally new cell type, the hybridoma. Thanks to genetic engineering, tobacco cells glow with a firefly's light, mice manufacture human antibodies, corn plants produce bacterial insecticide, and bacteria churn out human pharmaceuticals. In California, a man sues his doctor to recoup profits from cell lines derived from his cancerous spleen; throughout the world, laboratories abound with HeLa cells, descended from the cancerous cervix of a woman named Henrietta Lacks, who died in 1953, quite unaware that her cells would live on. Optics Carl Zeiss Inc. One Zeiss Dr. Thornwood, N.Y. 10594 Phone: (914) 747-1800 Product: DynaCELL workstation: Contact vendor for price Media Ventrex Laboratories 217 Read St. Portland, Maine 04103 Phone: (800) 341-0463 Products: PC-1 serum-free media for primary cell cultures: $20 (100 ml); $61 (2 units, 500 ml each). Powdered concentrate for 10 l: $396 HL-1 for non-anchorage dependent hybridomas: $7 (100 ml); $46 (2 units, 500 ml each). Powdered concentrate for 10 l:$231 CHO-1 complete media for Chinese hamster ovary cultures: $55 (2 units, 500 ml each). Powdered concentrate for 10 l:$228 Grow and freeze cells in cryoprotective medium, 10 vials, stored in liquid nitrogen for 1 year: $325/cell line Cytometers Becton-Dickinson Immunocytometry Systems P.O. Box 7375 Mountain View, Calif. 94039 Phone: (800) 223-8226 Products: FACStarPLUS: Single laser: $134,500; Dual laser: $198,800 FACScan (benchtop model): $99,500 Coulter Corp. 601 West 20th St. Hialeah, Fla. 33010 Phone: (800) 526-6932 Fax: (305) 883-6820 Products: Profile II, clinical flow cytometer: $85,000 Elite, clinical and research flow cytometer:$225,000 and up Meridian Instruments Inc. 2310 Science Parkway Okemos, Mich. 48864 Phone: (800) 247-8084 Fax: (517) 349-5967 Products: ACAS 570, attached cell cytometer: $200,000-$300,000 Magnetic Beads for Cell Sorting Advanced Magnetics Inc. 61 Mooney St. Cambridge, Mass. 02138-1038 Phone: (617) 497-2070 Fax: (617) 497-6927 Products: Various magnetic goat antibodies: $79-$89 (50 ml) BioMag-separator (permanent magnet, holds five 15 ml tubes and three 50 ml tubes for cell sorting): $295 Various magnetic binding proteins and lectins: $89 (5 ml) Laser Traps Cell Robotics Inc. 1285 B Trinity Dr. Los Alamos, N.Mex. 87544 Phone: (505) 662-5563 Fax: (505) 662-5781 Products: Laser Tweezers micromanipulator: $25,000 - $40,000.. To add Laser Tweezers computer control and image processing module: $25,000 - $50,000 Cell Robotics Workstation I: $200,000 - $250,000 Microspheres Verax Corp. 6 Etna Rd. Lebanon, N.H. 03766 Phone: (603) 448-4445 Fax: (603) 448-5681 Product: VX-100 Collagen Microspheres: $495 per kit This week (from December 9 through December 13), researchers are gathering in San Diego for a celebration of the cell--the 1990 symposium of the American Society for Cell Biology. The meeting of the Bethesda, Md.-based society reflects the explosive growth of the field, exploring intracellular processes (including chromosome replication, microtubule sliding, membrane channel behavior, and protein folding) and intercellular processes (such as cell migration, cell-cell and cell-matrix interactions, and cell movement during development). Novel combinations of tools offer tantalizing glimpses into cellular function. Monoclonal antibodies linked to fluorescent molecules zero in on specific structures and track their functions. Marriages of microscopes, image-enhancing software, and video cameras provide never-before-seen views. Consider, for example, the team of differential interference microscopy and video-enhanced imaging, which has "completely turned around microtubule biology," according to David Pennock, assistant professor of zoology at Miami University in Oxford, Ohio. "This pairing has allowed people to visualize single microtubules, only 2 nanometers in diameter, which is theoretically too small to be observed in the light microscope. But, somehow, digitizing the image allows one to see a single microtubule," says Pennock, who studies cytoskeletal movements of the protozoan Tetrahymena thermophila. The DynaCELL workstation, available from Carl Zeiss Inc. of Thornwood, N.Y., goes even further--this video microscopy system allows observation of cells in motion, which the company dubs "cell morphometry." Also in attendance at the meeting this week will be many of the manufacturers of the tools that make modern cell biology possible: tools that represent an eclectic mix of the old and the new. "Some techniques are 20, 30, even 50 years old, and have really not been superceded by anything new. There is still a great place in cell biology for classical techniques that have been proven," says James Whitman, president and director of research at Columbia, Md.-based Advanced Biotechnologies, referring to such old standards as light and electron microscopy, chromatography, centrifugation, and density gradient separation. Nuts And Bolts Dozens of companies supply the "nuts and bolts" of cell biology--growth media, media storage bags, growth factors and cytokines, cell lines, bioreactors, monitoring devices, and a wide variety of plastic culture vessels carved with nooks and crannies to encourage cell growth. And many companies have formed to support the burgeoning applications of cell biology. Biotech Research Laboratories Inc. of Rockville, Md., for example, takes a client's cells--from the American Type Culture Collection, in-house stocks, or the client's own source--and custom-cultures them, offering the expanded product in flasks, roller bottles, or wet or frozen concentrated pellets. The company also offers hybridoma services; it will fuse the cells that make monoclonal antibodies. Quality Biologics Inc., based in Camden, N.J., tests other companies' biopharmaceuticals for viral contamination. And several firms serve as consultants, wading through the dizzying array of bioreactor components to design the most cost-effective system to suit a particular client's project needs. Cell biology has come a long way since Hooke discovered cell walls more than 300 years ago, and the field has virtually exploded over the last two decades. Using sophisticated techniques and equipment, researchers can grow, sort, and manipulate cells to a degree unheard of just a few years ago. Nurturing Cells Bacterial cells are easy to culture because they thrive suspended in fermenters. But animal cells, which are more fragile and persnickity, present a dual technological challenge. For one thing, the media used predominantly until the early 1970s contained horse or fetal calf serum, or chick embryo extracts, the contents of which were not constant and were often unknown. "If there is serum in the media, you will have contaminants that will make it difficult to run experiments to determine the effect of certain substances, such as growth factors," says Hank Founds, senior vice president for technical operations at Ventrex Laboratories, headquartered in Portland, Maine. "If you want to produce a product to be used for therapeutic purposes from cells, [it would be advantageous to have] a well-defined media that doesn't vary from lot to lot." Hence, the advent of serum-free media. Typical serum-free media for mammalian cells contain amino acids, vitamins, salts, glucose, antibiotics to prevent bacterial growth, phenol red (the color of which is monitored to hold the pH around 7.4), and perhaps a growth factor specific to the cell type. The second challenge in culturing mammalian cells is to not harm them, yet mix things up sufficiently in bioreactors so that stagnant pools, or "microenvironments," do not develop. Researchers have come up with many innovative ways to cater to these useful but temperamental cells. For example, four dozen companies offer electromagnetic stirrers powered by rotating magnetic fields to keep things moving. Beads called microcarriers give cells something safe to adhere to during culturing. Founds says that the microcarriers manufactured by Ventrex "range from a core of glass or plastic coated with gelatin, to pure gelatin carriers." Verax Corp., based in Lebanon, N.H., offers collagen microspheres that provide the best of both types of environments: Suspendable cells nestle in the billions of tiny caverns that pervade the porous beads, while anchorage dependent cells attach to the outside surfaces. Some new products satisfy cells' need to cling as well as biologists' need to observe. A tissue culture plate insert offered by Wheaton, of Millville, N.J., is an inorganic membrane that is porous enough to allow ions, macromolecules, and nutrients to reach both the attached and free surfaces of cells. The membrane becomes transparent when wet, so a researcher armed with a microscope can see cells grow. And Meridian Instruments in Okemos, Mich., has developed a cover-glass chamber for cell culture that permits fluorescent image analysis, selective cell destruction, and even time-lapse videos of live cells. Sorting Cells Often a researcher needs to obtain a single type of cell from the many that make up a tissue. Sedimentation and centrifugation, older sorting techniques based on size and density, fail to distinguish cells of similar sizes and densities that are otherwise quite different. Another time-tested tool with wide clinical applications, yet some limitations for the researcher, is a Coulter counter, a device commonly used to rapidly count blood cells. The Coulter counter works on a simple theory, called the Coulter principle, developed by Wallace Coulter in the basement of his Chicago home in 1947: A very narrow stream of particles in suspension passes a detector, which counts them. "You place a probe in a suspension of cells, and it siphons a known volume of cells through a single opening, across which an electric field runs. Every time the field is broken by a cell coming through, [the Coulter counter] counts it," explains Miami University's Pennock. The Coulter principle is echoed in the more sophisticated technological descendant of the Coulter counter, the flow cytometer, developed around 1980. Flow cytometry both counts and differentiates between cell types--a process crucial in diagnosing leukemia, for example--by measuring the reflection of light off fluorescent markers on a cell's surface and internal structures. In the fluorescence activated cell sorter (FACS; pronounced "fax"), developed in the early 1980s by Leonard Hertzenberger and coworkers at Stanford University, cells labeled with antibodies bound to a fluorescent dye are suspended in saline and sent under pressure through a pipe only a few microns in diameter. Drops leaving the vibrating nozzle at its end are so small that each contains, at most, one cell. This single file of cells passes a laser, where a positive charge is generated if a cell doesn't fluoresce, and a negative charge is generated if it does. A strong electric field then deflects marked cells into one container, unmarked cells into another, and empty and overloaded droplets into a waste receptacle. A FACS is a major investment. Firms supplying these devices include the Hialeah, Fla.-based Coulter Corp. and Becton-Dickinson Immunocytometry Systems, of Mountain View, Calif. But it might be more prudent for potential buyers to wait for a "third-generation" particle sorting machine about to debut from Lawrence Livermore National Laboratory in California. Ger van den Engh, a senior scientist at Livermore, has invented an instrument that can exceed the 5,000 particles per second handled by current FACS devices. A limitation of flow cytometry is that the cells must be in suspension, a state that is unnatural for many cell types accustomed to life in a multicellular organism. This might not be important for clinical applications, such as leukemia diagnosis, but it can be a critical shortcoming in research. "There are cells, in culture or in tissues, that have to be in communication with other cells. If they are put into suspension, the researcher loses in- formation," says Noah Hadas, Meridian Instruments' vice president for marketing and sales. ACAS 570, a cell sorter designed by Meridian to overcome this hurdle, combines cytometry and microscopy technologies to offer astounding new capabilities. "Although the laser, photomultiplier tubes for detection, and optics are similar to those of flow cytometry, the difference is that cells are left adherent, in their natural state," Hadas says. Cells are grown on a culture plate 35 millimeters in diameter, lined with a thin plastic film. Using a microscope, the researcher scans the field for a cell of interest, then cuts it out with the laser, a protocol dubbed "the cookie cutter step" at Meridian. When the sheet is lifted up, the desired cell--virtually untouched--is left behind and can be used to clone a population of cells. Most exciting about ACAS 570 is a confocal optics accessory that allows for the viewing of spectacular optical sections and the measurement of ions, pH, or biochemical flux. About 70 of these cytometers have been sold worldwide since the device was introduced on the market in 1986. Researchers need not necessarily come up with thousands of dollars to sort their cells, however. An alternative or complement to a pricey FACS is magnetic separation, an approach pioneered in the last few years. Cells are incubated with monoclonal antibodies, washed, and then incubated with magnetic particles bearing antibodies that fit like puzzle pieces to the antibodies on the cells of interest. A magnet then separates the magnetically marked cells from the mixture. This approach is used in a new leukemia treatment that may obviate the need to find a compatible marrow donor. "You remove bone marrow from a patient and give him chemotherapy. Then mix magnetic beads with the bone marrow to remove cancerous cells with antibodies specific for the individual. Then reinject the cancer-free bone marrow back into the patient," says Steven Moss, president of Dynal Inc., a biotech firm based in Great Neck, N.Y. Recipes for magnetic particles vary among the handful of companies commercializing them. Dynal's Dynabeads are coated with polystyrene, to which proteins can be conjugated. BioMag particles, offered by Cambridge, Mass.-based Advanced Magnetics Inc., have an iron oxide core coated with a thin layer of silane, an organic, silicon-containing polymer, to which various chemical groups are attached. Dennis E. Vaccaro, vice president of marketing at Advanced Magnetics, suggests that BioMag particles be used before a FACS to concentrate cells that make up less than 1 percent of a sample, which are not easily sorted from the suspensions that flow through cytometers. The magnetic particles are too small to disrupt the FACS, but they save time and enhance sensitivity. Manipulating Cells Cell biology meets laser technology again in the exciting new field of optical trapping, in which a single or dual laser beam holds a cell in place, much like a ping-pong ball is held aloft by jets of air. "Researchers can trap and manipulate individual cells and chromosomes to a 1 micron accuracy. You can levitate cells, fuse cells, hold them very precisely, or even move organelles," says Ronald Lohrding, president of Cell Robotics Inc. in Los Alamos, N.Mex., a company spun off from Los Alamos National Laboratory to develop and commercialize a line of laser trapping modules, or "laser tweezers," invented by biophysicist Tudor Buican. Although relatively slow, laser tweezers will be valuable for "rare cell analysis," Lohrding predicts. For example, they might be used to pluck fetal cells from the maternal circulation, replacing more invasive prenatal protocols. Alternatively, they could be used to isolate stem cells from bone marrow, from which new tissue can be cultured. Plant protoplasts can be enticed to fuse in a laser trap once a cell is located, using a computer programmed to recognize certain molecular topographies. Laser trapping should prove particularly valuable for the needle-and-haystack job of spotting a hybridoma that produces a particular monoclonal antibody. "It gives biotechnologists the opportunity to pick cells out of billions, fuse them, clone the fused cells in a chamber, and observe if they produce biomolecules with the combination of properties that they want," Lohrding says. Today's cell biologists have an unprecedentedly wide variety of tools to choose from, enabling them to observe, culture, sort, and even manipulate cells. Perhaps these devices, coupled with scientists' creativity, will enable researchers to explore this intriguing unit of life even further. "We have a better understanding of cellular processes, but there are still lots of gaps in our knowledge of what takes place in cells," says Advanced Biotechnologies' Whitman. "For example, we don't know what really happens when a virus infects a cell, does what it does, and is then kicked back out of the cell. Or, particularly in developmental biology, we don't really understand the highly regulated cascade of signals from outside to inside cells. We know bits and pieces, but we do not know how they interact. We have a long way to go to understand the basic processes of cells." But the tools to do so may already be in existence. Ricki Lewis teaches biology at the State University of New York, Albany.

Special Report: Cell Biologists Combine Old And New Tools

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