Summing up the resounding cell culture industry view, Bruce Lehr, director of marketing at SAFC Biosciences (www.sigmaaldrich.com), says “the trend is continuing to take serum out of industrial processes.” That trend continues to push further up the pipeline, with a growing emphasis on serum-free, animal byproduct-free culture conditions in the cell line generation phase.
A major driver is to determine, as early in the development process as possible, whether a target molecule will be safe and effective in the clinical setting before devoting significant time and resources to optimizing the processes associated with producing the compound.
As a result, media suppliers, such as SAFC Biosciences, are developing serum-free and animal component-free media formulations that work well across different clones and cell lines to standardize these early-stage processes and enable production of sufficient quantities of compound to move forward into clinical testing. These formulations can later be tweaked to optimize growth and productivity of a particular clone.
Of increasing importance in both cell biology research and drug discovery is the desire to study the effects of compounds and various stimuli on cell function, physicochemical properties, and signaling pathways in a specific target cell or tissue. To understand what is happening in the liver, for example, researchers study the functional properties of hepatocytes. The broader use of many different types of cells, particularly in drug discovery, is increasing demand for media designed to optimize culture conditions for individual cell types.
Matching Media to Cells
“In cell biology, a key focus is to obtain information that is as close to what is happening in the organism of interest as possible,” says Marshall Kosovsky, Ph.D, technical products and programs manager at BD Biosciences (www.bdbiosciences.com). For growing hepatocytes, for example, BD offers a cell environment that includes culture media optimized for hepatocyte growth together with culture plates coated with BD Matrigel™ Matrix.
Dr. Kosovsky describes the BD Cell™ MAb media formulations, designed for use in the BD CELLine™ 1000 membrane-based device, as a system intended to maximize recombinant protein expression and high-yield Mab production. BD Biosciences’ cell environment systems include media optimized to promote the growth and differentiation of a variety of cell types together with extracellular matrix (ECM)-based growth substrates.
Dr. Kosovsky points to the study of angiogenesis as a prime example of how researchers are moving toward 3-D cell culture, allowing the study of endothelial cells in an environment that closely mimics blood vessel formation. The BD BioCoat™ Angiogenesis System facilitates the study of endothelial cell Invitrogen (www.invitrogen.com) is planning two key research product launches this month, including its first 3-D cell culture matrix to enable the growth of tissue-like structures, according to Martin Naley, vp of cell culture research. The second group of products will be additions to the company’s line of cell-specific media. The most recent addition to that line was a specialty media for growing bronchial epithelial cells.
“We clearly see that the trend to replace animal cell culture systems or immortalized cell lines by human cell culture system due to the higher significance of the experimental outcome gains more and more momentum,” says Luka Kadovic, market development manager, at PromoCell (www.promocell.com). “This is complemented by a steadily increasing demand for human cells that are used for establishing specialized cell-based models; for example, blood brain barrier model system, angiogenesis model system, skin model, etc.” Kadovic identifies endothelial cells, keratinocytes, fibroblasts, and melanocytes as being in high demand.
“In addition, we noticed an increasing demand for lymphatic endothelial cells dedicated for basic research on lymph angiogenesis, homeostasis, wound healing, and drug development.” PromoCell used cell type-specific markers to identify and isolate highly pure cell populations. Specializing in human primary cell culture, the company’s product portfolio includes a range of human primary cells and optimized media as well as cytokines, antibodies, and reagents required for cell and tissue culture applications.
Replacing Animal-based Materials
Novozymes (www.novozymes.com) continues to grow internally as well as through acquisition. In July, it acquired Delta Biotechnology—a producer of recombinant human transferrin (DeltaFerrin™) and recombinant human albumin (albucult™), both animal component-free products intended for use in the preparation of cell- and tissue-based therapeutics—from the Sanofi-Aventis group (en.sanofi-aventis.com) to form the Novozymes Delta subsidiary (biopharmaceuticals.novozymes.com).
Novozymes recently completed its acquisition of Australia-based GroPep, which distributes its LONG®R3IGF-1 human therapeutic insulin alternative through the Sigma Aldrich SAFC division.
“Beginning in 2007, Novozymes will position itself as a key provider of biopharmaceutical ingredients,” says Dermot Pearson, Ph.D., commercial operations manager at Novozymes Delta. “The availability of a range of animal-free growth factors and supplements will make it easier for companies to move away from serum-containing media.”
As stem cells move closer to near-to-patient use and regulatory issues loom larger, Novozymes is targeting its albucult recombinant albumin product to the stem cell culture market. “Our customers are looking for animal-free products that are consistent from batch to batch,” according to Dr. Pearson.
Even how providers prepare and supply cell culture media to their customers is changing with disposable options, such as HyClone’s (www.hyclone.com) media preparation vessel, the single-use mixer (S.U.M.) that complements the company’s single-use bioreactor (S.U.B.) technology, offering traditional stirred tank suspension in a disposable bag system.
The S.U.M. and S.U.B. will a have capacity of up to 1,000 liters working volume in 2007. The use of HyClone’s disposable Powdertainer™ powdered media handling vessel enables direct media delivery to the S.U.M., offering a fully disposable flowpath from powder media packaging to the bioreactor.
All of these advances represent Hyclone’s “drive toward integrating disposable systems with animal component-free media and reagents in response to market demands,” says Brandon Pence, bioproduction market manager. The combination of disposable systems and animal component-free media and reagents provides regulatory and validation benefits.
Other recent product additions that exemplify this effort include an expanded line of animal component-free media, such as HyQ® CDM4MAb™, a chemically defined medium for producing Mabs in hybridoma and myeloma cells, and HyQ SFM4MegaVir™ for the production of viral vaccines in cell culture.
Focusing on the changes taking place in production strategies for influenza vaccines and the transition away from egg-based systems to cell culture methods, Hyclone introduced the HyQSphere™ microcarrier line that provides an adherent, animal component-free surface for the culture of vaccine-producing cells. The microcarriers allow for vaccine manufacture in traditional bioreactor systems, simplifying scale-up and facilitating large-scale production.
Standardized Media for Production
In the bioproduction market, new product development has been driven by customers’ desire for reduced variability and simplification of culture conditions to facilitate process design and reduce the complexity of downstream purification. Invitrogen introduced a new insect cell culture medium, Sf-900™ III SFM, which is a low-hydrolysate, protein-free, animal origin-free medium optimized for growth of Spodoptera frugiperda cells and for the large-scale production of recombinant proteins expressed using the baculovirus expression vector system.
“Feedback from our customers asked for reduced variability in hydrolysates to help with experimental design,” says Linh Hoang, M.D., Ph.D., business area manager for cell culture bioproduction.
To enhance CHO cell culture, Invitrogen also launched the CD-OptiCHO medium, an animal origin-free, chemically defined product.
Irvine Scientific (www.irvinesci.com) recently introduced IS CHO FEED-CD, a chemically defined, animal component-free feed medium for fed-batch CHO cultures developed to improve the growth and productivity of multiple CHO cell lines. While it was designed to be used specifically with the company’s chemically defined media, it can also be used with a variety of other CHO media.
“We are seeing an increased interest in feed media for fed-batch cultures,” says Sandy Parten, senior product manager for industrial cell culture at Irvine. Some companies are even moving away from using perfusion and toward fed-batch culture systems. IS CHO FEED-CD can be used for both chemically defined processes and those that include hydrolysates. The company’s Optimized Hydrolysate Mixture for CHO Cells, as well as its soy and yeast hydrolysates are all ultrafiltered for improved consistency.
Irvine’s main focus is developing and manufacturing proprietary and custom media for industrial cell culture. The company’s R&D group recently presented a poster describing the development of a chemically defined feed medium for a specific CHO cell line in three weeks using spent media analysis, analyzing what nutrients are deficient and what are present in abundance in the spent media.
“Cell titer increased 98% using the custom feed,” says Parten. Two different feed media prototypes were derived from the spent media analysis—one was based on simple replacement and the other on a depletion rate based strategy,” she explains. “The feed prototype based on the depletion rate strategy led to a greater improvement in performance than the simple replacement based prototype.”
Irvine has noted increased interest from potential European and Asian customers and a continuing focus on speed of delivery by customers worldwide. Irvine’s Custom Media Program and Express Media Service offer media in four to six weeks and one to two weeks, respectively.
All Eyes on Stem Cells
StemCell Technologies (www.stemcell.com) has followed the evolution of stem cell research, early on bringing to the market standardized media and reagents designed for cultivating hematopoietic stem cells and associated assays for characterizing and enumerating stem and progenitor cell populations. The company’s emphasis continues to focus on developing standardized conditions for stem cell culture based on optimized media and reagents that yield consistent, reproducible results both within and across laboratories.
Now, as the variety of stem cells being used for research and potential clinical applications has grown to include nonhematopoietic stem cells, including mesenchymal and neural stem cells, Sharon Louis, Ph.D., senior scientist at StemCell Technologies, once again sees the need for standardization of basic research tools and culture conditions, so researchers can compare data produced in different labs. This includes the need for standardized media optimized for these new stem cell populations and for standardized assays to identify and quantify stem cells and facilitate their isolation without contamination by other cell types.
“We have moved into the mesenchymal and neural stem cell field, as well as offering products designed for use with endothelial and breast stem cells and human and mouse embryonic stem cells,” explains Dr. Louis. She notes a growing demand for ESC research products.
According to Dr. Louis, even though the existence of neural stem cells was reported nearly 15 years ago, there is still not one standardized medium and set of protocols for growing neural stem cell colonies and measuring neural stem and progenitor cells. Various labs continue to use different combinations of cytokines and growth factors. “Standardization of the tools will bring the needed robustness and stringency to the early stages of stem cell research,” she adds.
PAA (www.paa.com) offers a range of media and reagents for the growth and differentiation of stem cells, including hematopoietic, mesenchymal, neuronal, and embryonic stem cells.
“It is important for labs to agree on the same platform for cell cultivation,” points out Susan Donath, product manager at Miltenyi Biotec (www.miltenyibiotec.com), emphasizing the need for standardized solutions. She describes a similar requirement for defined cytokine and growth factor preparations for use in cell culture and differentiation.
Different labs may generate contradictory results regarding the differentiation potential of a stem cell line, for example, but it might be unclear whether those differing results are due to the cell line itself or the culture conditions. The growing importance of being able to compare results between laboratories is contributing to the need for chemically defined media in stem cell research.
“The big trend at the moment is the move away from undefined media formulations,” says Erik Schneider, head of clinical product development. There has been a gap between efforts to enrich for cells with potential therapeutic applications and their subsequent use in a clinical environment, Schneider adds.
This regulatory gap is driving the push for sera- and animal component-free, defined media. Some alternative formulations to replace sera, such as soybean hydrolysates, which are plant-based and fulfill the need for an animal-free product, are under development.
Miltenyi Biotec is building on its cell separation systems designed for isolating defined cell populations and is offering culture media optimized for the cultivation and differentiation of enriched stem cell populations. Examples include the company’s MACS® stem cell media for cultivating hematopoietic and nonhematopoietic stem cell populations, including mesenchymal stem cells, and media designed for the expansion and isolation of antigen-specific T-cell populations. Media for the expansion of different populations of immune cells are also in development.
Simplifying Cell Culture
To overcome the problem of variability in culture conditions for growing embryonic stem cells (ESCs) due to the use of a feeder cell layer to support the survival and maintenance of the cells in an undifferentiated state, scientists at the Scripps Research Institute screened a library of synthetic small molecules and identified a compound they called pluripotin that supports self renewal of mouse ESCs and maintains their pluripotency in the presence of only standard basal cell culture medium. The compound also appears to enhance the growth of human ESCs but is not sufficient to maintain their pluripotency. The researchers are screening libraries of analogs of this class of synthetic pyrimidines to identify a compound that could fulfill that role.
For applications in stem cell biology and tissue engineering research, BD Biosciences recently introduced BD BioCoat Matrigel Matrix plates for ES cell culture that support stable proliferation of undifferentiated human embryonic stem cells in culture without the need for a cell feeder layer.
Mediatech (www.cellgro.com) is working with its customers in the area of ESC research to take culture environments with traditionally high concentrations of serum and bring them down to low levels of animal byproducts. Under construction and slated to begin operations by mid-2007 is Mediatech’s new 100,000-sq-ft facility that will include areas designated as animal byproduct-free.
Highlighting the exciting advances being made in regenerative medicine and the use of tissue engineering to grow replacement organs, Jim DeOlden, president of Mediatech, describes the emerging need for large quantities of serum-free, animal component-free, and well-defined media and the chemical supplements needed to replace the function of serum proteins and help advance this work into human trials and clinical applications.
In the area of large volume, sterile solutions delivery and sterile filling technology, Mediatech maintains exclusive rights in North America to David S. Smith’s (www.davidssmith.com) sterile packaging technology. The company manufactures disposable bags with a sterility assurance level (SAL) of 1x10-8. Within the next six months, bags will be available with a SAL down to 1x10-12, according to John Elliot, executive vp at Mediatech.
Millipore’s (www.millipore.com) acquisition of Chemicon/Serologicals allowed the company to merge its expertise in the membrane-based cell culture market with the ability to produce media and reagents for cell growth and characterization.
“We are focusing a lot of our efforts around stem cell biology,” says Michelle Greene, marketing director at Millipore. She describes growing demand for products that serve both the animal and human stem cell markets and support the cultivation of both adult and embryonic stem cells.
“It is imperative to understand how stem cells grow because, in general, the knowledge lags well behind traditional cell types,” Greene adds. “Therefore, the trend in the stem cell research market is moving toward serum-free, animal component-free, and chemically defined media.”
She describes an increasing recognition in the stem cell marketplace that a shift away from animal-based materials lies on the horizon as stem cell culture applications move closer to clinical testing.
Earlier this year, Millipore released Esgro Complete™ serum-free media for growing mouse embryonic stem cells in a feeder-free defined system intended for use in studying stem cell differentiation and for making transgenic animals. The company also introduced ReNcell™ immortalized adult human neural somatic stem cells as well as rat and mouse adult neural stem cells media for maintaining and differentiating the cells, and antibody-based kits for cell characterization. To meet the growing demand in adipogenesis research, Millipore also offers rat mesenchymal stem cells.
Adult Stem Cells
Focusing on the adult stem cell market, R&D Systems(www.rndsystems.com) developed the StemXVivo™ media product line based on MSC-based media and serum-free NSC Base Media with defined mixtures of supplements and growth factors.
The MSC-based media can be used for the expansion of mesenchymal stem cells and for differentiating MSCs into adipocytes, chondrocytes, and osteocytes. The NSC-based media can be used for the expansion and differentiation of neural stem cells. In addition, R&D has developed formulations optimized for culturing dendritic cells. A T-cell medium is due on the market in early 2007. The company uses primary stem cells for media development and evaluation of differentiation efficiency.
“Validation of the raw materials is very important for a stem cell culture media product,” says Jessie Ni, PhD, assistant director of the stem cell and antibody application groups at R&D Systems. “Lot-to-lot variation can be huge,” she adds.
The newest addition to SAFC Biosciences’ Stemline™ family of stem cell media is a neural stem cell expansion medium for use in the R&D of cell therapy applications. In keeping with the trend toward serum-free environments, this new medium along with other Stemline hematopoietic, dendritic, and T-cell media are serum-free and contain no growth factors or antibiotics.
Lin Hoang, of Invitrogen, notes that in the bioproduction area, “our customers are inviting us in more and more to work with them to optimize media and culture conditions.”
Invitrogen is investing in SimCell technology, a robotic, fully automated, microfluidics-based system developed by BioProcessors (www.bioprocessors.com) that uses miniaturized reactors—MicroBioreactors, or SimCells—operated in parallel to simulate full-scale culture conditions and enable high-throughput process development.
Each SimCell holds a 150–1,000-µL volume. An accompanying software system manages the experiments and monitors an array of parameters related to the culture environment, cell behavior, the addition of nutrients, metabolite production, and product yield. Invitrogen offers this service to allow customers to experiment with culture conditions in a scaled-down system and optimize media for a particular cell line.
SAFC Bioscience’s Cell Express service offers high-throughput cell line and media screening. Clones producing high levels of the desired protein are retained, while laser energy is used to destroy surrounding, less productive cells. The goal is to match cell lines with an optimal media faster and to achieve higher productivity of a cell line to facilitate the initial characterization studies.