Better Understanding Of Cell's Life Eases Culturing

The term "cell culture" brings to mind several cliches--"if it ain't broke, don't fix it" and "the more things change, the more they remain the same," to name a few. While the requirements of cells growing in culture haven't changed over the years, better understanding of the precise combinations of nutrients, growth factors, attachment factors, and the like necessary for a cell's life apart from the organism has eased cell culture, always an integral part of the cell biology laboratory. "Lots of companies come out with media or reagents for this or that, and make a big splash, but they're all basically derivatives of traditional products," says Hayden Coon, a former National Institutes of Health re-searcher who is the founder of Human Cell Therapies Inc. of Chebeague Island, Maine. Advanced Biotechnologies Inc. Columbia, MD American Qualex Antibodies La Mirada, CA American Type Culture Collection (ATCC) Rockville, MD BioWhittaker Inc. Walkersville, MD Boehringer Mannheim Corp. Indianapolis, IN Cell Systems Corp. Seattle, WA Cellco Inc. Germantown, MD Cellpro Inc. Bothall, WA Celox Laboratories Hopkins, MN Circle No. 216 on Reader Service Card Electron Microscopy Sciences Fort Washington, PA Circle No. 220 on Reader Service Card Fluka Chemical Corp. Ronkonkoma, NY Circle No. 221 on Reader Service Card Gallard-Schlesinger Industries Inc. Carle Place, NY Circle No. 222 on Reader Service Card Human Cell Therapies Inc. Chebeague Island, ME HyClone Laboratories Inc. Logan, UT Circle No. 224 on Reader Service Card ICN Biomedicals Inc. Costa Mesa, CA Circle No. 225 on Reader Service Card Intergen Co. Purchase, NY Circle No. 226 on Reader Service Card Irvine Scientific, Inc. Santa Ana, CA Circle No. 227 on Reader Service Card JRH Biosciences Lenexa, KS Circle No. 228 on Reader Service Card Life Technologies Inc. Gaithersburg, MD Circle No. 229 on Reader Service Card Mallinckrodt Specialty Chemicals Co. Chesterfield, MO Circle No. 230 on Reader Service Card Pharmacia Biotech Inc. Piscataway, NJ Promega Corp. Madison, WI Protein Polymer Technologies Inc. San Diego, CA Research Diagnostics Inc. Flanders, NJ Research Organics Inc. Cleveland, OH Rockland Immunochemicals Gilbertsville, PA Sigma Chemical Co. St. Louis, MO Thomas Scientific Swedesboro, NJ Unisyn Corp. Tustin, CA Wako BioProducts Richmond, VA Worthington Biochemical Corp. Freehold, NJ "Some companies call new products proprietary, but they're just developed slightly beyond the original. They are variations on a standard theme. But as long as it meets the goals of what we're doing, we're satisfied," asserts Coon, whose biotech startup is planning to develop pancreatic islets and other cell implants for treating human disease. Although suppliers of cell-culture media and reagents often rely on established formulations developed in the 1950s, such as the popular Dulbecco's modified eagle medium, they have managed to keep pace with the many new discoveries in cell biology, offering an assortment of individual reagents, kits, and custom services for culturing a variety of cell types. Cell culture has evolved along with molecular biology advances in general, and to recombinant DNA technology in particular. Before the late 1970s, cells were coaxed to thrive in culture so that their secreted natural products could be collected. With the advent of recombinant DNA technology, researchers and then pharmaceutical manufacturers could endow cells with new synthetic capabilities, ushering in the age of "biological" therapeutics. "True tissue-culture people are trying to maintain cultures with growth factors, cytokines, and such. There's no change there from years past. But there are now more people using cell culture as a tool to investigate what cells do," comments Shari Harrison, product manager for a line of cell- proliferation and cell-death kits from Indianapolis-based Boehringer Mannheim Corp. "Molecular biology, cell biology, immunology, and biochemistry--once separate disciplines--are now totally merged," she says, referring to the diverse scientists who now use the kits. The future of the field, according to many researchers, will be in implants. Unlike the past uses of cell culture as an incubator to churn out a needed biochemical, cell-implant technology aims to nurture cells for the purpose of transferring them to a human body, where their secreted products will prove therapeutic. "I envision, perhaps 20 years from now, a series of ampules, on shelves, with cell therapies administered by a nurse to anyone who has a complaint," Coon predicts. Cell-culture supply companies will not have to shift gears much, if at all, to accommodate the fledgling field. "Cell- therapy technology may have a different goal than traditional cell culture, but the companies that already manufacture media can just follow the recipes of any research group, according to what types of cells they plan to implant," Coon adds. Most researchers use secondary cell cultures, grown from cell lines obtained from another researcher or from a facility such as the American Type Culture Collection of Rockville, Md. For a researcher to start a primary culture--one directly from an organism--cells must first be disassociated. This might involve using a protease (such as trypsin or collagenase) and a chelating agent to chemically break apart adherent cells, and then a mechanical disruptor to further separate them. Cell types are then grouped by physical differences such as weight, size, or cell-surface topography. Cells are given enough space, in an appropriate biochemical milieu, not only to survive, but also to divide and specialize. Epithelial cells in culture form sheets; muscle cells join into pulsating groups; neurons sprout extensions; fibroblasts churn out collagen. A culture medium typically includes nutrients (amino acids, a carbohydrate such as glucose or galactose, and vitamins), inorganic salts (of magnesium, sodium, potassium, calcium, phosphate, chloride, sulfate, and bicarbonate), antibiotics, and perhaps a fungicide such as amphotericin B. Also included are chemicals for the culturist's benefit--phenol red to indicate pH, and trypan blue to distinguish dead from living cells. Until recently, another basic component of cell-culture medium was serum, usually from fetal calf or horse. Serum provides a wealth of benefits to cells, including nutrients, hormones, attachment factors, and carrier proteins for water- insoluble compounds. Serum also neutralizes some toxins and buffers the culture medium. But using serum has a drawback--it adds a whole set of unknowns, possibly including infectious agents such as viruses and mycoplasma. "Actually, using serum is okay, if your goal is to grow cells. But if your goal is to grow cells and understand their physiology, you need another kind of approach," according to Coon. That approach is to use low-serum or serum-free media, which means that what leaves with the serum must be replaced with whatever mix of growth factors, hormones, and cytokines a particular cell type prefers. Another reason to minimize serum usage, or at least to track its geographic origin, is the recent outbreak of bovine spongiform encephalopathy (BSE), or "mad cow disease," in England. Concern over contaminated serum prompted the Food and Drug Administration to alert cell- culture reagent manufacturers, on Dec. 17, 1993, that they shouldn't use bovine materials from England and other European nations where BSE is seen. "Now the industry is looking for serum sources from only a few places--the U.S., Canada, New Zealand, and Australia," says Richard Wilkinson, senior director of research and product development at HyClone Laboratories Inc. of Logan, Utah. Cost is also a factor in avoiding serum. HyClone's FetalClone serum substitutes are about half the price of serum-based media. "We picked the name to convince the public that it looks like bovine serum-based media, but it's not," notes Wilkinson. FetalClone I is suited to hybridomas, II to Chinese hamster ovary (CHO) cells, and III to fibroblasts. HyClone also offers a Complete Culture Media (CCM) line that is serum-free. Says Lucy Cherbas, staff scientist at Indiana University, Bloomington: "We grow some of our Drosophila cell lines on HyClone's serum-free CCM 3 medium, which is cheaper than mixing up a serum-based medium. They won't tell us what's in it, but we have no need to know--the cells grow!" CCM 3 is specialized for insect cells. Other CCM products target hybridomas (CCM 1), human fibroblasts (CCM 2), keratinocytes (CCM 4), and CHO cells (CCM 5). Another vendor of serum-free insect media is JRH Biosciences of Lenexa, Kan., which offers kits consisting of complete, assembled media called EX-CELL 400, 401, and 405. In addition to Drosophila, a mainstay of genetics, another big market for insect cell-culture media is the baculovirus expression system that uses ovarian (Sf-9) cells of the pupa of the armyworm Spodoptera frugiperda. St. Louis-based Sigma Chemical Co. also offers serum-free media kits tailored to specific cell types. "Each kit is composed of basal medium plus necessary supplements," says Joel White, technical services representative in cell culture. The keratinocyte medium kit (KMK-1), for example, includes basal medium, bovine insulin, bovine pituitary extract, human recombinant epidermal growth factor, gentamicin, amphotericin B, and hydrocortisone. Other kits are useful for culturing fibroblasts or endothelial cells. "The kits save researchers not only time, but they save economically," White adds. "If a researcher were to buy all of the growth factors needed to supplement a cell culture, it would be very expensive. He or she would be trapped into buying larger quantities than needed. The kit puts it all together quite conveniently." Patrick Chang, product group manager at BioWhittaker Inc. of Walkersville, Md., ticks off what he considers the top four "of about 20" reasons to avoid serum. "The biggest reason is that animal blood may contain viruses, some that we have no assay for. If you are running a pharmaceutical or biotech company and are manufacturing a drug or a biological, using cells in culture with serum-based media is taking a large risk. Second is the cost, and third is the consistency. When a lot of media runs out, and a researcher gets some from a new lot, there can be large differences in content. Finally, because of the defined nature of serum-free medium, we know exactly what's in it. Parts of serum may be unknown, such as growth factors we don't yet know about." BioWhittaker offers a new serum-free product, called Ultra- MEM. "Most serum based media require 5 to 10 percent serum to work well. Our new formulation requires less than 10 percent," reports Chang. The company also custom-manufactures medium. Human Cell Therapies' Coon is one satisfied customer. "We gave BioWhittaker a list, and they did a magnificent job. They did it well, fast, and we got good results," he says. For researchers choosing to buy single cell-culture components and cook up their own defined medium, most vendors offering kits also sell individual items, such as growth factors, antibiotics, and amino acids. Some companies sell combinations of reagents, such as JRH Bioscience's EX-CELL 900, which is a premixed preparation of bovine insulin, transferrin, and selenium, the three most commonly used media supplements for mammalian cell culture. The trio of chemicals enhances proliferation of many cell types, while decreasing serum needs from 10 percent to 2 percent, according to company literature. Intergen Co., located in Purchase, N.Y., doesn't itself offer kits, because it has so much business supplying individual cell culture reagents to companies that then package them in this way, according to Michael Budnick, manager of technical/marketing services. For example, the company is the largest supplier in the world of bovine albumin, a plasma protein. "It's being used more and more in biotechnology and pharmaceutical production where customers are trying to get away from fetal bovine serum and use a more defined medium. You can reduce or eliminate fetal bovine serum if you use albumin, transferrin, insulin, and growth factors," says Budnick. Albumin has numerous roles in cell culture, including stabilizing, providing protein, and protecting against oxygen damage. Several new products are keeping cell-culture technology on track with cell biology advances, ranging from individual reagents, to kits, to entire bioreactor systems. Switching from serum-based medium to serum-free medium stresses cells. The finicky cells might not attach to the new surface, or may not proliferate. As cells that can't adapt die, the nature of the cell population subtly shifts, altering experimental results. San Diego-based Protein Polymer Technologies Inc. offers ProNectin F, which can ease this tough transition for cells. The product is a protein polymer built of repeats of a tripeptide that is part of fibronectin, a natural attachment factor, encapsulated in a polymer coating. The protein portion is synthesized in recombinant bacteria. "ProNectin F allows the rapid passage of cells from serum-based to serum-free media without extensive weaning protocols," explains Mary Forrest, product manager. "It's ready to use, which appeals to industry, as opposed to culturing with lots of reagents," she adds. Cellco Inc. of Germantown, Md., for example, uses ProNectin F in its CELLMAX Artificial Capillary Cell Culture Systems, designed for growing mammalian cells for research and pharmaceutical applications. Eventually, the device will be used for ex vivo cellular therapy, in which a patient's cells are collected, cultured in the system, altered genetically, allowed to proliferate with the correction, then returned to the patient, where their bolstered capabilities should correct a disease state. "Cellco and its collaborators have confirmed that ProNectin F provides for im-proved cell adhesion and growth while enhancing the biocompatibility of our products," says R. William Lynn, president and CEO of Cellco. "These are very critical considerations when working with therapeutically useful cells such as endothelial and bone marrow stroma." Boehringer Mannheim offers several kits that embrace a more molecular approach to monitoring the life, reproduction, and death of a cell. "It used to be that, to study cell proliferation, you'd put yourself at a microscope and count cells. Now there are several ways to do it," notes product manager Harrison. "The MTT and XTT Cell Proliferation Kits measure metabolic activity in the cell, using tetrazolium salts in a nonradioactive colorimetric assay," Boehringer technical services scientist Sharon Mountain explains. The salts are oxidized by cellular and mitochondrial enzymes, forming a dye. "But there is a danger of not getting perfect data if cells show increased metabolism, but are not proliferating. So the user can look at a true indication of proliferation, which is DNA synthesis," adds product manager Harrison. She is referring to researchers working with the company's cell- proliferation kits, based on incorporation of bromodeoxyuridine (BrdU), a base analog, into replicating DNA. A fluorescently tagged antibody to BrdU highlights cells that have incorporated the analog, indicating new cells and therefore measuring proliferation. Keeping pace with what Harrison calls an "exploding" interest in programmed cell death (apoptosis), Boehringer Mannheim offers two products, the Cell Death Detection Kit (introduced in February) and the Cellular DNA Fragmentation Kit (introduced in May). Programmed cell death is an active process that sends a cell through a characteristic sequence of events. At first, blebs form from the cell membrane, the cytoplasm condenses, and the nuclear membrane breaks down. The next step is where the test intervenes. The self- destructing cell produces an endonuclease that cuts up its DNA- -but only at sites where the nucleic acid is exposed, and not protected by the histone proteins that form part of a chromosome's structure. After the endonuclease snips the double helix into 180-base-pair stretches of DNA hidden in histones, the cell shatters, and scavenging cells absorb its remains. This dance of death is distinct from other ways that cells die. The products can help researchers distinguish between necrosis, which is the passive breakdown of structures as a cell dies, and apoptosis, which is an active process. Because these routes to cell death differ, they may underlie different disease states. The Cell Death Detection test is an immunoassay that utilizes anti-histone antibodies to detect the characteristically sized histone-DNA complexes the endonuclease liberates. The Cellular DNA Fragmentation test uses BrdU to quantitate the DNA pieces. The promising future of cell-implant technology has led some cell culturists to reexamine the state of their art, because the need for the technology is compelling. "About 6,000 pancreases are donated a year, two or three are needed to cure one patient of diabetes, and there are 1.4 million diabetics. This is an area where amplified cells in culture could make a great impact," says Human Therapies' Coon. But cell-implant technology won't be feasible until cell culturists learn much more about the individual requirements of different cell types, says Randal A. Goffe, chief technical officer and vice president at Unisyn Corp., headquartered in Tustin, Calif. "The reason hepatocytes have taken so long to work in an artificial liver, and artificial pancreases and livers have never succeeded clinically, is because cell- culture systems aren't efficient enough," he explains. "At the current metabolic efficiency, you need massive numbers of cells to treat one person," Goffe adds. "But if the cells were performing at their peak efficiency, you would need only a small device, and a realistic number of cells. At that point, we will attain clinical efficiency. Cell culture is the key to moving forward in cellular therapy. We must discover the nuances of how to treat cells. No one has optimized cell- culture and bioreactor technology. We must go back to basics, not just throw cells into something and hope for the best." Researchers at Unisyn are taking that "tear it down and build it back up" approach to perfect a product in development called Unigrow, which is a hollow-fiber bioreactor. Cells grow in narrow spaces between hollow fibers bundled into glass or plastic cylinders. The setup mimics an animal's circulatory system, with fibers delivering oxygen and nutrients, and taking away wastes and secreted substances. Goffe is currently attacking what he calls "the oxygen paradox"the fact that cells need oxygen, but too much of it or too close contact is lethal. "In cell culture, when you increase the oxygen, you kill the cells," he says. "From an engineering standpoint, how do we design a system so that doesn't happen? That's what we're involved in." He is evaluating several hemoglobin-based formulations as oxygen deliverers in the Unigrow bioreactor-in-progress. "It only works if you separate the cells from the hemoglobin, and that's where a hollow-fiber bioreactor comes in," he points out. "Like in the body, the hemoglobin products bind carbon dioxide where metabolic wastes build up and drops it, picking up oxygen, in regions where oxygen's partial pressure is higher." Goffe adds that the system works on any cell type, even the notoriously fussy hybridomas. Goffe is particularly interested in hematopoietic stem cells. He founded Bothall, Wash.-based Cellpro Inc., a company specializing in stem-cell separation. Stem-cell culture could be invaluable in treating many disorders, because these cells give rise to mature blood cells. Nurturing stem cells in vitro presents quite a challenge, because they often progress through several distinct stages before attaining their final differentiated state. He notes: "It is important to control the microenvironment for hematopoietic stem cells, and to supply nutrients, oxygen, and a specialized medium for long-term expansion. To do this, you must go back to first principles--strip the system down to simple components and rebuild." Ricki Lewis is a textbook author and freelance science writer based in Scotia, N.Y.

Better Understanding Of Cell's Life Eases Culturing

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