Postmodern Culture: Maximizing Cell Culture Output At Every Level

Date: July 5, 1999Products for High-Volume Tissue Culture The Automation Partnership's CELLMATETM Automated Cell Culture Processing System Mammalian cell culture has finally come into its own as a way to produce cells, proteins, or sub-cellular components in large scale. Recent advances in cell culture systems are allowing scientists to better circumvent problems associated with bacterial systems such as incomplete protein expression, toxin contamination, and incompatibility. Today's wide array of flasks, bottles, reactors, and controllers allow even the smallest lab to scale up quickly and economically. The system of choice depends on a number of factors: lab resources, type and quantity of the desired product, the frequency and duration of the project, and the cell type used. This article focuses on tools to scale up cell culture for the occasional research study and even full-scale industrial production. The table summarizes the various culture systems and manufacturers. Tools for cell harvesting will be addressed in a future issue of LabConsumer. Although one cell type grows well in a flask, it may fail to thrive in larger and more complex arrangements. Simple, linear scale-up is, by and large, uncomplicated for flask- and roller bottle-based systems, but conditions must nearly always be redefined for high-density capabilities. Consult with each company's technical service representative for advice on which medium is best for your culture system. Suspension cells are the simplest to scale up for all systems. Flasks and stir bottles work best for these cells, especially biomass harvest. Monitoring of pH, O2 saturation, and waste products can be done directly with a minimum of disturbance to the system. Image supplied by Unisyn Technologies Adherent cells adapt well to roller bottle-, hollow fiber-, and microcarrier-based systems. Whole-cell harvest is facile with roller bottles, while hollow fibers are better for secreted products. Microcarriers are used in both biomass and secreted product harvests. Adapting your culture to serum-free media is highly advantageous for simplifying purification of protein products. Specialized media formulations have been developed for most common cell types. Recently, researchers have taken to transfecting cell lines with growth factor genes to simplify transition to high-density and serum-free conditions. As the culture ages, attrition via apoptosis becomes an issue. Proteases and other undesirable "death products" reduce product yield and complicate purification. Hybridoma cells expressing high levels of bcl-2 gene have been shown to resist programmed cell death.1 Unfortunately, apoptotic pathways are multifaceted, so increased bcl-2 expression alone won't reduce apoptosis in all cultures. While the life sciences have their share of excellent technicians, not many of them will attend to culture demands around the clock, handle large volumes of liquids, work with lethal pathogens, and consistently produce quality results on a 7/24 basis. To this end, many large biotech companies employ robots in the manufacture of antibodies, viruses, and other bioproducts. Accordingly, the space and financial considerations restrict them to the industrial setting. The Automation Partnership's CELLMATETM is an example of a robotic system for processing roller bottles and T flasks from initial seeding to harvesting. The company works with customers to design and implement a system exactly suited to production needs. For the research lab doing a pilot study or infrequent modest scale-up, the simplest solution is to use larger chambers. Increasing the number of large flasks quickly becomes inefficient and time consuming if you don't hire more support. A better solution is to increase the surface area of the culture with systems like those from Nunc, Corning, or Panacea Solutions. Nunclon Cell FactoriesTM are low-profile, disposable, polystyrene, ventable chambers that come in stacks of one, two, 10, and 40. Inoculation, feeding, and harvest are straightforward due to the innovative design of the connected plates. No special training is needed to use the system, making startup a snap. Cells can be visualized directly in the chambers via an inverted microscope. Nunc® also offers a robotic manipulator for large industrial facilities. New Brunswick Scientific's Packed Bed Perfusion Bioreactor diagram Bioreactor diagram The CellCube from Corning Science Products is an easily expandable system for growing adherent cells in all levels of biomass, viral, and soluble biomolecule production. These disposable polystyrene modules hold 3.5 l of media and contain 25 parallel plates for a total growing area of 21,000 cm2 per cube, expandable up to 340,000 cm2 (the 4/100 stack). The interlinkable cubes stand on one corner with media entering the bottom and exiting the top. The digital controller features in-line monitoring of perfusion, pH, dO2, and temperature and is Windows 95TM compatible. The Wave BioreactorTM from Panacea Solutions International employs an adjustable-speed rocking platform and electric air pump to gently aerate the culture while keeping shear forces low. Simply seed the cells, seal, and incubate. Smaller cultures and rocking platforms will fit in a standard incubator. Rockers and CELLBAGTM bioreactors are available in a range of sizes, from 2 l to 100 l. Roller bottles are used in every conceivable application. A good starting point for small labs with periodic scale-up needs, they are also being used for large-scale industrial production. Because the cultures are seeded and maintained in a manner similar to flasks, typically no additional training is necessary. Small racks fit inside standard incubators, eliminating the need for additional capital expenditures. Roller bottles come in a number of configurations: plastic, glass, pleated, flat, vented, or solid. Glass can be sterilized and reused, whereas different plastics and coatings optimize growth for an assortment of cell types. Pleats increase the effective growth surface, thereby increasing product yield without additional space requirements. Vented caps are used for culture in a CO2 environment, while solid caps are best for culturing in a warm room or unregulated incubator. Companies supplying roller bottles and accessories are outlined in the table. The need to augment biomass and secreted product yield led scientists to develop a system that could allow cells to live in increased density. Stir flasks and hollow fiber systems were first to be developed and enjoy a history of success. But the field continues to evolve as new systems are developed for increasingly diverse needs. Rotary bioreactors use rolling action to keep cells well perfused, akin to roller bottles. In order to create a high-density environment, the culture chamber is separated from the feeder chamber by a semipermeable membrane. This allows media to be changed without disturbing the cells. Using this principle, the rotating action in Synthecon's Rotary Cell Culture System (RCCS) creates a microgravity environment, virtually eliminating shear forces. This allows the cell to shift resources from damage control to establishing relationships with other cells, mimicking the complex three-dimensional (3-D) matrices found in vivo. According to the company, this is the only known system to produce organized, 3-D, differentiated, tissue-density cocultures outside the body. Reactor vessels come in sizes ranging from 10 ml to 500 ml. The Heraeus miniPERM bioreactor combines an autoclavable outer nutrient container and a disposable inner bioreactor chamber. The user chooses the appropriate molecular weight cut-off membrane for the desired product. Its small size allows it to fit inside standard incubators. Densities greater than 107 cells per ml and product yields of 160 mg in four weeks are common, according to the company. Historically, the largest cultures of cells have been achieved in fermenter-type systems. Used in settings from small labs to big industry, the low cost, ease of scale-up, and modest space requirements make this a good choice for many applications. Suspension cells are most direct to scale up in this system. Once the parameters for achieving maximum product have been delineated, it's a simple matter to grow and harvest the culture. In-line monitors for pH, gas saturation, and metabolites are available from most of the suppliers. Adherent cells pose more of a challenge. Some can be "suspension-adapted," while others simply refuse to "get with the program." Cells of the latter persuasion usually need microcarrier beads as a support. Particles come in two forms: solid and porous. Solid beads are the most manageable for biomass harvest, while porous beads are better suited for secreted or lysate products. Other matrices hold beads stationary, creating a solid bed through which media are perfused. One disadvantage of solid beads is that the cells are constantly crashing into each other and experiencing shear force, resulting in higher levels of necrosis. Lysosomes and proteases are released from the dying cells, decreasing the yield and quality of the product. Recognizing this, companies have developed stirrers and beads that minimize collisions and shearing. Porous beads have historically suffered from heterogeneity of pore size, thereby limiting the actual numbers of cells/particle in culture. In response, manufacturers have developed new materials with a more uniform pore. Amersham Pharmacia (AP) Biotech offers microcarriers and fluid-bed reactors. Cytopore I beads are optimized for CHO-type cells, while Cytopore II is for adherent cells requiring higher surface-charge density. Cytoline I beads are suited for resilient cells requiring high circulation rates. The low-density Cytoline II carrier is optimized for shear-sensitive cells such as hybridomas needing slower circulation. AP Biotech has designed the Cytopilot fluid-bed system perfusion reactor for use with its Cytoline beads. B. Braun Biotech's Biostat® Bioreactor Porous ImmobaSil microbeads produced by Ashby Scientific are available in different shapes and sizes for easy adaptation to your particular culture vessel. They are gas permeable, allowing culture densities to reach 3x106/ml for maximum product yield. CYTOSTIR® from Kimble-Kontes is a line of double-sidearm bioreactors in nine sizes, from 100 ml to 36 l. The large, height-adjustable stirring blades are constructed of Teflon to minimize cell adhesion and facilitate cleaning. All components are steam-autoclavable. The ProCulture DynaLift system by Corning® facilitates perfusion and reduces shear effects by using an extended paddle, side baffles, and bottom contours. It is available in a range of sizes, from 125 ml to 36 l. New Brunswick Scientific's CelliGen Plus® is a highly flexible system for culture of virtually all eukaryotic cell lines. Features include a double screen impeller for increased O2 saturation, interactive four-gas control, internal ring sparger, five programmable pumps, computer interface for system control and data logging, and four-channel recorder output. The unit may be used either as a stir tank or fibrous-bed system, as conditions dictate. While it is currently sold as a 1.4 l reactor, custom systems are available for larger needs. Applikon® offers a full line of stir tanks, from 2.3 l bench systems to 10,000 l production units. Pumps, probes, controllers, and software are also available for all units. Borosilicate glass vessels are available up to 20 l and can be fitted with lip-sealed or magnetically coupled stirrers. Stainless steel BioClaveTM vessels are designed for moderate to large-scale production and feature a flush-mounted longitudinal sight glass as well as a choice of lip-seal or magnetic stirrers. The SuperSpinner from B. Braun Biotech is an entry-level stir flask that accommodates 500 and 1000 ml cultures and features a patented, bubble-free aeration/agitation system. The Biostat® series of stir vessels handles culture sizes from 50 ml to 10 l and include complete ready-to-use systems and systems that integrate preexisting components. Techne UK offers a complete line of stir flasks in volumes up to 5 l. Designed with a stirring rod rather than paddles, they simplify cleaning and autoclaving by eliminating rotating bearings. The unique stirring action creates vertical and horizontal flow in a gentle spiral throughout the culture. Its line of programmable stirring platforms features the SOFTSTART acceleration/deceleration control to reduce cell damage from excessive turbulence. Quark Enterprises provides a full range of bioreactors including its SPINGRO® flasks for high-density culture. These borosilicate stir flasks range from 100 ml to 36 l and feature Teflon spin paddles, side vents for probes and easy sampling, and jacketed models for use with a recirculating water bath. All models are completely autoclavable. Wheaton Science Products offers scale-up systems for all levels of production. Its Magna-Flex® Spinner Flasks have bulb-shaped, flex-type glass impellers for use with microbeads. A removable stainless steel pin immobilizes the impeller to prevent cell damage during handling. Available in a range of sizes from 125 ml to 8 l, they are fully autoclavable. Also available are the Cell Optimizer SystemTM for determination of optimum culture conditions prior to scale-up, and the OverdriveTM for economical industry-level production up to 45 l. Cellex Biosciences' AcuSyst hollow fiber reactor schematic An innovative alternative to the stirred tank is the airlift bioreactor. The reactor has no moving blades to create shear forces, which some mammalian and hybridoma cells are particularly sensitive to. Media perfuse from the top while oxygen enters through the bottom, creating a near-ideal mixing environment. One difficulty lies in large-volume scale-up. The need for a small cross-section-to-height ratio means that a 30 l fermenter requires a 3 m ceiling height. Kimble-Kontes offers the CYTOLIFT® glass bioreactor with an effective volume of 580 ml. It is easily cleaned and fully autoclavable for consistent performance and long life. A glass jacket is standard on all models. Other features include a check valve to prevent backflow in case of pressure drop, vent, infusion and effluent ports, plus three ports for pH, foam level, and dO2 probes. Late in the 1980s, a major breakthrough in cell culture came with the adaptation of hollow fiber technology from renal dialysis systems. Media are flushed down the center of the fiber while cells grow in the interfiber space. Media and small molecular weight solutes freely cross the fiber wall, while product and cells are trapped on the other side. The design of the system makes it difficult to use for biomass production, but is well suited for soluble products. Researchers (and mice!) will be glad to know the days of tedious ascites harvesting are all but gone since the advent of hollow fiber systems. Today's per-day cost to maintain a colony of mice approaches the cost of spending a day in the finest five-star hotels. And the mice don't even get a private room or personal trainer! Now with hollow fiber culture, once the hybridoma is isolated, simply seed the "mouse" and let it crank out milligram quantities of antibodies. Cellex Biosciences makes hollow fiber reactors for all levels of production. The AcuSyst-XCELL® is designed for large-scale production of secreted proteins, producing 60 to 200 grams of protein per month. Its AcuSyst miniMaxTM is a flexible research scale benchtop bioreactor capable of producing up to 10 g of protein per month. For single-use or pilot studies of a few weeks' duration, the economical ResCu-PrimerTM produces up to 200 mg per month with a choice of hollow fiber and ceramic matrices. The Unisyn Cell-Pharm® MicroMouseTM is a disposable system with a footprint of 1.5 ft2, fitting inside a standard lab incubator. It is capable of producing up to 250 mg of monoclonal antibodies per month for three months. The Cell-Pharm System 100TM is a freestanding benchtop system with a disposable flowpath with yields of up to 400 mg/month. For larger settings, Unisyn produces four other Cell-Pharm models for extended mg to g scale production. The TECNOMOUSE by Integra Biosciences is a modular rack system with five separate cassette chambers. Up to five different cell lines can be cultivated for up to 30 weeks, each producing 200 mg of antibodies per month. The integrated gas supply and online monitoring capabilities help to control culture conditions. The latest design has come in the form of Kinetic Biosystems' CBR 2000 centrifugal bioreactor. Designed for industrial production, it can achieve densities up to 10,000 times greater than stirred tank bioreactors, according to the company. Media are fed in through the axle, then forced to the outside by the rotating action where they enter the reaction chamber. Cells are held in suspension by opposing centrifugal force with perfusion. Waste products are removed through the axle and sampled 10 times per hour. Real-time analysis of growth and production parameters means that any perturbation can be adjusted quickly. The end result is increased product yield and quality. Each chamber is capable of producing 1x1016 cells with each rotor holding three chambers. With so many choices available for even the most modest production needs, there is no good reason not to kick production into high gear and get modern! Products for High-Volume Tissue Culture Y. Itoh et al., "Overexpression of bcl-2, apoptosis suppressing gene: prolonged viable culture period of hybridoma and enhanced antibody production." Biotechnology and Bioengineering, 48:188-122,1995. Additional reading: Basic Cell Culture Protocols Methods in Molecular Biology, Vol. 75, Totowa, NJ, Humana Press, Inc., 1997. The author can be contacted at gmeisenholder@the-scientist.com.

Postmodern Culture: Maximizing Cell Culture Output At Every Level

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