INSIGHTS on the Elements of Cell Culture

Things to Consider When Starting or Running a Cell Culture Laboratory

Stem Cell Culture Media / HyClone HyCell-STEM and HyCell-STEM-FF / Thermo Fisher Scientific www.thermoscientific.com

Collangenase/Neutral Protease Blends STEMzyme™ 1 & 2 / Worthington Biochemical www.worthington-biochem.com

Bioreactor Flask & Animal Component-Free, Chemically-Defined Medium / CELLine™ & CHO Growth A / WHEATON & Irvine Scientific www.wheaton.com & www.irvinesci.com

The cell culture marketplace is diverse and wide-ranging, crossing through and over many industries. It includes medical testing and diagnostics, toxicology testing, drug discovery, forensics, environmental testing, cell-based assays, foods, beverages, and supporting services for pharmaceuticals and biotechnology.

One can dice and slice cell culture in dozens of ways, most commonly by industry, application, cell type, culture method, or end-product. This article takes a product-based approach, with the major categories being media, culture ware, and large equipment. This allows inclusion of the broadest range of cell types, culture conditions, and industries.

Media—the Lifeblood of Cells

The prime differentiator between cell culture and most other scientific disciplines is that cells are living entities that need to be fed and nurtured. While biologists have made tremendous progress in designing new cell lines through gene splicing, experts recognize cell culture as the single most important factor in generating high cell performance and productivity. Where animal cell culture has traditionally employed variable serum-based media, scientists are slowly abandoning this “magic sauce” for products with greater definition.

“Users are looking for speed and consistency and off-the-shelf usability in mediums and reagents,” says Jessie H.-T. Ni, PhD, chief scientific officer at Irvine Scientific (Santa Ana, CA). “They want products ready-made, out of the box.”

Ni compares options for cell culture media to serving prepared foods vs. cooking from scratch. The latter may be tastier and more artful, but it takes time and scientists are often in a hurry.

That’s not to say all off-the-shelf media are the same. Scientists employ different ready-formulated products for different stages of culture, or enddeliverables. Similarly for stem cell culture, they will use one medium for expansion and another to induce differentiation. “But even then, they don’t want to have to add growth factors,” Ni explains. “They may not expect optimized products in every case, but they do desire all components to be contained in one package. It doesn’t have to be the best as long as it’s consistent.”

Nathan Allen, product marketing manager for media and serum at Thermo Fisher Scientific (Pittsburg, PA), notes that while industrial cell culture increasingly relies on defined media, academics are not as interested in switching. “Most research continues to use classical media products with serum supplementation, which are widely available, well known, and have broad applicability. The major exception is stem cell culture, where many researchers want more defined conditions.” While media development and optimization do occur in academic settings, most researchers prefer off-the-shelf media because media optimization is difficult and time-consuming. “Most researchers do not have the patience or time to develop new media,” Allen adds. “The exception occurs with specific cell lines that are not well served by existing media.”

Thus, the burden of media design and feed strategies, at least for research-grade cell culture, has fallen from end-users onto media suppliers. Yet for large cultures, where output may be valued at millions of dollars, media and feed are optimized through an iterative process that continues to production scale.

Monitoring and feedback in large cultures

Cell Culture Shelving and Incubation Products / Gelf ™ Caron / www.caronproducts.com“We are entering a new era of cell culture media and feed design,” says Bill Whitford, senior manager, cell culture and bioprocessing at Thermo Fisher Scientific (Logan, UT). “Blending existing formulations to create a more optimized basal growth media is no longer sufficient. Nor is adding a lump dosage of a metabolite cocktail into an expanding culture.”

New analytical techniques and methods of cell characterization provide novel data on molecular systems involved in cell culture production platforms. “These range from new high-throughput and content cellular analysis to dissociation techniques and label-free mass spectrometry.” This supports the rapid analysis of ambient components from trace metabolites to high molecular weight products. Second, new methods to employ this data are emerging that enable manufacturing process developers to characterize culture progression, product expression, and bioprocessing.

“These tools not only shed light on the primary mechanisms involved, but indicate markers of key pathway activity during the extent of the culture,” Whitford adds. Prominent among these is systems biology, which describes the dynamic relationship between cell phenotype, gene expression, and intracellular metabolite levels.

Other technologies are emerging to support either the closed fluid circuit (in situ sampling with bypass) or cell-free sampling of bioreactors and at-line real-time measurement of metabolite levels and other critical process parameters.

These techniques include novel approaches to monitoring such as disposable, calibrated probes, techniques for measuring dielectric properties of cells, and plasmon resonance to characterize cell productivity.

Pre-coated Cell Culture Products / GrowCoat™ Sarstedt / www.sarstedt.comUntreated Polystyrene Culture Microplates / Porvair Sciences www.porvair-sciences.com

 Culture ware: Where the cells live

Cells will grow in a wide variety of containers, provided conditions are right. Culture vessels range from uncontrolled microtiter plates to 10,000-liter bioreactors and everything in between.

Microtiter formats are attractive for screening cells before plating them out or expanding them in t-flasks, shaker bottles, or other types of culture ware. What holds true for biology in general applies to cell culture.

“As the field progresses, investigators seek to use less media, reagents, and cells to extract the information they need,” says Grazie Mendonsa, PhD, marketing manager at Midwest Scientific (St. Louis, MO). Ninety-six well plates are common in cell culture, but many protocols call for plates with as few as six wells.

Some plate products are coated to promote adherence, but that depends on the cell line. Immortalized cells (hybridomas) have no issues growing on untreated surfaces. Primary cells often need assistance, typically from coated extracellular matrix proteins or optomechanical treatments, or they will not attach and grow.

“Primary cells are more difficult to work with, and they only divide about 25 times before they die,” Mendonsa explains. Still, scientists are intensely interested in primary cells because they most closely resemble “natural” somatic cells.

Automation has not taken traditionally manual cell and tissue operations by storm as it has in other areas of life science research. Automated liquid handlers and robotic plate handlers are more likely to be used in sample and standards preparation and for treating cells once they are plated. Microorganism cultures employ automation to ensure precise metering of organisms into assays. “But cell isolation is still mostly a manual operation,” Mendonsa says.

To Ken Ludwig, business manager for cell culture at Corning Life Sciences (Tewksbury, MA), customers most often look for a complete package of media, culture ware, and specialized surfaces that encourage the attachment and growth for a variety of cells. “The way to improve productivity is to get things right from the beginning. Helping with a selection of vessels, media, and surfaces starts culture labs in the right direction, without the need for them to try a lot of different combinations.”

With its late 2012 acquisition of Becton Dickinson’s Discovery Labware business, Corning became arguably the largest vendor of cell culture ware. Perhaps the single most important aspect of this deal was broadening Corning’s offerings for growth surfaces. With the purchase of Mediatech in 2011, Corning gained “unique play” in the cell culture marketplace, offering cellspecific media as well as t-flasks, multiwell plates, and specialty culture vessels, according to Ludwig.

Pall (Port Washington, NY) has not historically been a cell culture company. But with its acquisition of Mountain View, CA-based MicroReactor Technologies, the company found itself among the global leaders in microbioreactor products and technology. Microbioreactors are small, controllable culture vessels used in biopharmaceutical process development and other types of small-scale cell culture R&D. Other companies in this space include Eppendorf (through its acquisition of German firm Dasgip), Applikon, and TAP Biosystems.

Stem Cell Medium / Essential 8™ Life Technologies / www.lifetechnologies.com

Automated Cell Counter / TC20 Bio-Rad / www.bio-rad.com

Compared with microtiter plates used ubiquitously in early-stage cell culture work, microbioreactors offer much lower throughput but with the ability to control culture conditions. Users of the Pall products can work with pH, dissolved oxygen, and temperature. Other systems control stirring, gassing, and other factors relevant to cell growth and/or protein expression. The Pall microbioreactors offer total working volumes of 10 mL in 24 wells, with an optimal volume of about 7 mL.

Through the use of incubators, microplate systems can control somewhat for temperature and gases, but precise control is impossible and temperature edge effects are common. “Users must frequently make decisions based on results of one poorly controlled sample,” says Loe Hubbard, bioreactor applications manager at Pall.

Microbioreactors bridge the scale between shakenwell microtiter plates and larger flasks and bioreactors, explains Hubbard. “Shaken well” refers to the method of agitation for microplate-based cultures.

Microbioreactors are ideal for further investigating culture conditions that have been screened in the more highly parallel but less sophisticated plate-based experiments. As such, the two formats are complementary, not competing. Cells, media, feed, and culture conditions selected at the microbioreactor scale are then scaled to larger bioreactors.

Microbioreactors are a kind of trade-off of scale. They are somewhere between microplates and low liter-scale in terms of setting up experiments and data analysis. Their versatility and controllability ranges between those of the two other formats as well. But, as Hubbard says, “They allow scientists to use both microplates and liter-scale bioreactors more intelligently.”

Freezing Medium / STEM CELLBANKER® / AMSBIO / www.amsbio.comPharmaceutical biotechnology seems to be a natural market for microbioreactors, but according to Hubbard this was not where microreactor technology took root. “Foods, forensics, and environmental were first, and most of those applications involved microbial fermentations.” Biopharmaceuticals, which mostly employ animal cells, are actually a new market for Pall’s microbioreactors, even though the company has been involved in large-scale biotech cell culture for many years.

On the horizon: Stem cell culture

Stem cell culture skills differ significantly from those of traditional cell culture. Stem cells are ultrasensitive: they grow as colonies, not as single cell monolayers or in suspension; they are not cultured in the presence of antibiotics; and they need to be carefully passaged to maintain pluripotency and to avoid random differentiation and genomic instability.

Chris Armstrong, PhD, VP of primary and stem cell systems at Life Technologies (Madison, WI), sees the role of media vendors as simplifying culture through products that are simpler, easier to use, and highly defined. Traditionally, stem cells were grown atop a layer of “feeder” cells that provide nutrients and keep the stem cells healthy. A recent trend, toward feeder-free culture, involves chemically defined culture media to control variability. “Growing stem cells is very different from growing Chinese hamster ovary cells,” Armstrong says.

CO2 Incubator CB 53 / BINDER www.binder-world.com

CO2 Incubator / Heracell 150i / Thermo Fisher Scientific www.thermoscientific.com

Optimizing stem cell culture media demands an evolution of media development away from art toward more robust, “industrialized” products that perform consistently.

Defined media, where ingredients are characterized to low percentages, instill greater confidence in those working with, expanding, and differentiating stem cells.

In addition to allowing greater control over research-stage stem cells, defined media opens the door to clinical applications and translational research, a goal of top stem cell media suppliers and scientists. Several of Life Technologies’ media have received FDA class 2 approval, which lowers the regulatory burden for researchers as they move toward clinical studies.

The shift to chemically defined stem cell media parallels the move to similar growth techniques for cells used in biopharmaceutical research, development, and manufacturing. And similarly, it reduces the regulatory burden for products that eventually wind up in patients.

One of Life Technologies’ products, Essential 8™ media, is based on the work of Prof. James Thompson, at the University of Wisconsin, that reduces the number of ingredients for stem cell culture media to just eight.

Differences in culture ware between traditional and stem cell culture are more subtle than for media and feed. While both use similar formats (flasks, Petri dishes, microtiter plates), coatings that encourage attachment to culture ware are designed specifically to prevent premature differentiation and maintain pluripotency and genomic stability. Again, the focus is on simple, chemically defined substances. One recent Life Technologies product launch, based on a single protein (vitronectin), replaces more complex, less well-defined basal matrix materials.

As stem cells enter human testing and receive regulatory approval, the scale at which culture takes place will expand—or perhaps explode is a better term. Production will need to become less hands-on, more reliable, and quality-driven. Robotics and automated liquid handling systems will be essential components of scale-up, as they are today for routine stem cell culture. Several key automation vendors, among them Hamilton and TAP, produce liquid handling systems that are being validated for large-scale stem cell cultures.

Capital equipment: Maintaining control, providing protection

Incubators

Uwe Ross, president of Binder USA, sees cell and tissue culture evolving under pressure from systems biology and personalized medicine. “We are very close to figuring out how cells work and how to outmaneuver cancer mechanisms by manipulating signaling pathways,” he tells Lab Manager. Going the final mile in this effort will require “a lot more research” in systems that more closely simulate physiologic conditions versus the “rough environment we can now provide inside an incubator.”

Ross explains that cell growth chambers once focused mainly on maintaining physiologic temperature. “Then we learned that wasn’t good enough, so we added humidity and monitoring and mechanisms to provide nutrition. Then we added pH, carbon dioxide, and oxygen. We will arrive at even more parameters that more closely simulate what goes on within the organism.”

The driving force behind further efforts will be the explosion in research, development, and clinical applications of cell- and tissuebased therapies, including organ regeneration.

But Ross warns that regulatory approvals for such treatments will be difficult to obtain because these processes are not up to standards for scale, safety, and robustness. Medical applications will require a complete rethinking of entire product classes such as CO2 incubators, he says. A major hurdle will be the introduction of clean-room incubators with appropriate isolation and safety features.

“While it’s not possible to have factory-sized incubators, they will be larger, perhaps the size of a freezer, with compartments that separate your kidney from my kidney.”

The prime factor dominating these discussions is the avoidance and elimination of contamination. Ross cites the many sources of errant microbes within a cell culture environment: media, flasks, pipettes, gloves, workers, and especially surfaces. “Everything is a huge contamination risk.” Several apparently punctilious cell culture studies have been questioned because researchers could not rule out the possibility of contamination.

Contamination avoidance and elimination are two strategies that Binder and other top vendors have been following for some time. Ross claims that his company’s incubators have reduced surface area by a factor of three while eliminating or minimizing weldings, fittings, and corners where bacteria love to grow. Binder incubators feature a push-button sterilization conducted for two hours at 180ºC. This strategy automates sterilization and practically eliminates manual cleaning.

Class II Biological Safety Cabinet / Purifier® Logic®+ / LABCONCO / www.labconco.comBiosafety cabinets

Biosafety cabinets provide varying degrees of protection to product, personnel, and environment. Class I cabinets provide personnel and environmental protection, but because their design is open-front with negative pressure, they provide no significant protection for products. Class II cabinets are vertical laminar-flow devices that circulate HEPAfiltered air through the work space. These units provide all three modes of protection. Class III cabinets behave similarly but are completely enclosed and require working through glove ports.

“The level of protection you need is based on the cells and whether your process generates toxic gases or other substances,” observes Bill Peters, marketing director at NuAire (Plymouth, MN). More precisely, the protection level required depends on the toxicity of the cells or process and their quantity. For example, users might take more precautions with large quantities of tuberculosis bacteria than with very small quantities of a more pathogenic but easily contained virus. Lethal agents like Ebola virus are always subject to the most stringent containment.

What should potential purchasers consider before buying a biosafety cabinet? Peters suggests that users first review performance specifications to ensure that they meet current and anticipated needs. “They should also consider human interaction factors, ergonomics, functionality, and user-friendliness, and not just from a control standpoint. These are pieces of equipment that are in daily use.”

Next comes cleanability. Biosafety cabinets and incubators are growth chambers which, not coincidentally, are found to be extremely hospitable by many types of organisms. “Everything inside has the potential for things to grow in or on them.”

While biosafety cabinets tend to be trouble-free, failures occasionally occur. Peters therefore adds reliability and service to his list of factors to consider.