Editorial Article
Wednesday March 03, 2010
by Laura Lane
The landscape of cell culture looks much more vibrant these days. Gone is the bland horizon of a few traditional formulations. Now that scene most resembles a multicolored tableau of options that suit a multitude of uses and cell types. While classic reagents and tools still pervade laboratory shelves, the newest products provide the wherewithal to dig deeper into the biology of tumors, tackle the world of stem cells and improve production capacity.
Overall, the products introduced during the last few years represent unprecedented convenience for customers who need specialized media. In the past, researchers purchased basic media, added supplements and then sterilized their homebrews on their own. These days, “it’s all ready to go,” says Stephen Chang, chief scientific officer of Stemgent.
“Before it was like cooking, you combine a bunch of ingredients,” Chang says. “Now you can buy that media for specific cell types.”
One size does not fit all
With that capability, attitudes have changed. Media has been taken out of the shadows as the spotlight reveals its pertinence.
“For years, cell culture media was thought to be just media,” says Robin Rothrock, director of product line strategy at ATCC. “As we think more and more about cells and the microenvironments in which they grow, the contribution of cell culture medium is perhaps much more appreciated now than ever before.”
For example, ATCC recently introduced a product line, called Primary Cell Solutions™, designed to provide researchers with a full system approach for culturing primary cells. Each primary cell type is matched to an optimized complete growth medium, comprised of a cell-specific basal medium and key growth factors packaged in a ready-to-use kit.
“We want to provide researchers with a complete solution to guarantee their success,” Rothrock says.
Stem cell researchers are also enjoying the increased convenience and choice. Driven by the introduction of induced pluripotent stem cells, the field has exploded, creating a market for tools geared toward supporting the very unique needs of stem cells. Companies have responded by developing media and reagents that not only allow stem cells to flourish in culture, but have also freed cultures from relying on undefined factors from feeder cells.
Companies like Sigma-Aldrich and Stemgent offer xeno-free media for stem cells. The products are designed to be used without feeder cells, helping researchers to draw conclusions without questioning the effect of unknown components. In addition, xeno-free media opens the way to opportunities in the clinical arena.
“It takes a lot of research to come up with the necessary components to go into these products,” says George Sitterley, market segment manager for research cell culture at Sigma-Aldrich. “Feeder layers are actually kicking out growth factors, and we need to identify them.”
Sigma-Aldrich offers products for different types of stem cells, including mesenchymal, hematopoietic, dendritic, neural and epidermal. The company’s studies have shown that the media allows the cells to retain pluripotency and differentiate into the entire range of cells normally found.
Stemgent’s stem cell media, called NutriStem™, contains ingredients to preserve the pluripotency of human embryonic stem cells and induced pluripotent stem cells for at least 20 passages, with a normal karyotype and all the necessary biomarkers. In addition, the cells retain the capacity to differentiate into cells of the ectoderm, endoderm and mesoderm both in vitro and in vivo.
Synthetic Serum
Options for media that is free of animal components has widened across the board. Researchers require serum-free media in order to observe cells in environments in which each and every component is accounted for. Biomanufacturers can’t pass regulatory muster with the threat of prions and other contaminants that may linger amidst manufactured proteins and other biological therapies. Overall, serum-free media brings consistency and reproducibility to the table.
“Serum and biological materials have been the mainstay of cell culture for years and years,” says Linda Dohrman Hladik, marketing director of discovery biologicals for BD Biosciences. “More and more, there’s a need to reduce the amount of sera to better understand cell biology with a more defined cell culture system.
“The more you understand about what you’re putting in the culture, the more you understand about the cells itself,” Hladik says.
Suppliers have delivered. They've painstakingly studied and pulled apart the components of serum, pinpointing practically every essential nutrient necessary to mimic the conditions in a serum-free environment. In addition, several companies produce the media in facilities created solely for the production of serum-free media.
BD Biosciences will inaugurate their animal-free facility in July 2010. Called the AF2TM, which stands for animal-free and antibiotic-free, the stand-alone plant "will control raw materials to the tertiary level, setting a new standard for safety and quality for cGMP cell culture media and supplements," says Chuck Velnoskey, market development manager, advanced bioprocessing for BD Biosciences. Such media can then be used for conducting early research, aiding in transitioning the outcome toward clinical uses.
“Even researchers who might not be as concerned as manufacturers would know that when you go from research to manufacturing you must change the components of media,” Velnoskey says. “That can be challenging.”
BD has formulated its Select CD1000 media for flexibility. The components of this serum-free media are present at concentrations that allow enough nourishment for cells, but are also “optimized to accept further supplementation without creating a toxic situation that’s detrimental,” he says.
PAA Laboratories is promising the upmost consistency with its new ADCF powdered media for which the company has constructed a new manufacturing facility. Several companies already offer powdered media. PAA is producing its product using updated technologies to ensure improved homogeneity, optimal particle distribution and problem-free solubility.
“We knew the manufacturing capabilities in the world were getting old and outdated,” says Robin Goodreau, sales manager at PAA Laboratories. “We thought we could offer better technologies to make a more consistent product.”
The new equipment, based on process analytical technology, closely monitors the milling and blending process by keeping tabs on the temperature to prevent degradation. Controlling moisture levels prevents clumping. An air classifier catches oversized particles, which are sent back for more grinding. The result is true intra- and inter-batch consistency of particles less than 250 um.
The layered look
Media is only one part of the equation to successfully culturing cells. The surfaces of plates strongly influence the state of cells. To manipulate the cells for your purposes, you choose different types of coatings to layer onto the plates. Some are designed for production convenience. Others are developed to control the growth and development of cells.
“With advanced cell culture surfaces, you can achieve as good of a yield as with serum,” BDs Hladik says. “Our focus is to develop surface characteristics with a number of different chemistries that will function the same way as a biological mimetic.”
BD Biosciences offers BD PureCoat as a non-biological coating. It comes in two varieties, amine and carboxyl surfaces. Both are designed to boost cells proliferation, help cells to recover from freezing and improve rates of transfection.
One of Greiner Bio-One’s surface chemistries is Advanced TC™, a non-biological polymer modification designed specifically for culturing sensitive cells, such as primary cells or cells cultivated under restricted growth conditions such as serum-free or serum-reduced. Plated on the Advanced TC™ surface, the cells exhibit good proliferation rates and can retain their native morphology.
“We see a trend in non-biological coatings,” says Lara Marchetti, R&D product manager at Greiner, explaining that customers appreciate the advantage of the increased shelf life, room temperature storage, sterility and animal-free components.
Biologically-based coatings still play a big role within the cell culture arena, especially when you need to mimic the in vivo environment as closely as possible. In the case of biological coatings, such as Greiner’s Cellcoat™, the proteins parallel the extracellular matrix of tissues to which cells attach in the body.
You can choose the coating that most suits how you’ll be using the cells. For example, if you want to support differentiation in stem cells, you might plate them on a biological surface. To preserve their pluripotency, we would recommend choosing a non-biological surface. “There’s always the optimal surface for each type of cell,” Marchetti says.
In addition, think about how tightly the cells will stick to a coating. If you’ll be using the plates and cells for high throughput screening or high content assays, cells must attach well to survive a washing step. Otherwise, you’ll lose too many cells.
“With Cellcoat™ and Advanced TC™, the cells adhere and interact with the surface or coating,” says Rainer Heller, R&D product manager of high throughput screening at Greiner. “There’s a strong attachment so the washing step doesn’t result in a loss of cells.”
Third Dimensional Digs
Certain coatings introduce the possibility of three dimensional growth, thus drawing even closer to the in vivo environment.
“The classical 2D plates would never get as close to in vivo-like conditions as 3D culture systems,” says Sven Muehlfriedel, R&D product manager of cell culture at Greiner. “Real 3D growth of cells, and hence most native culture conditions may be achieved by utilizing specific devices, such as ThinCert™ cell culture inserts with porous membranes in combination with specific 3D matrices.”
Sigma-Aldrich provides a selection of 3D matrices. Their synthetic product, HydroMatrix™ Peptide Hydrogel, provides a scaffold of crosslinked peptides that cells can use as a base. Researchers can alter the nature of the 3D apparatus by changing the concentration of the gel. A biologically-based product, MaxGel provides a 3D matrix using proteins of the extracellular matrix.
“The collagen, fibronectin or other components affect what goes on in the cells when they bind,” Sitterley says.
Taking a new approach is InfiniteBio, which recently introduced its 3D Cell Culture NanoCulture® Plate System. Instead of the synthetically-produced or biological elements, the NanoCulture® system provides a nano-scaffold built on thin film, which is at the bottom of plates.
“Nanotechnology helps to make the pattern on such a small scale, which cells like,” says Ana Lazic, scientific project director at InfiniteBio.
In fact, Lazic explains, gene expression differs when the cells grow in a 3D environment. “The results from a 3D cell culture screening system will correlate with [those of] in vivo conditions,” she says. “That means you can screen compounds using 3D cell based asays and get in vivo-like drug responses earlier, before animal studies.”