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A place for my cells
July 2013
by Randall Willis  |  Email the author


In 1981, comedian George Carlin gave us his insights on our seemingly endless need for storage.  
"That's all you need in life, a little place for your stuff. That's what your house is, a place to keep your stuff while you go out and get … more stuff! Sometimes you gotta move, gotta get a bigger house. Why? No room for your stuff anymore."  
With seminal technological advances such as the development of induced pluripotent stem cells (iPSCs), the potential for stem cell technology has never been greater. Whether used as research tools to generate biofactories or models of human disease (which you can read about in our August 2013 issue) or as regenerative medicines or vaccine therapeutics (which you can read about in our September issue), stem cells offer researchers and clinicians a wealth of new opportunities.
But this wealth comes with a price tag, as companies and research organizations try to determine how best to grow and store the myriad cell lines for future use.  
Cache cowed?  
According to a report from GBI Research last July, the growth in biorepositories has been dramatic since the 1970s, with increases of 42 percent and 36 percent in the 1990s and 2000s, respectively. Changes to research funding budgets, however, significantly threatens this growth at a time when demand may be at its highest.
In 2011, Jimmie Vaught and colleagues at the National Cancer Institute's (NCI) Office of Biorepositories and Biospecimen Research expressed their concern about the threat to future cancer research coming from the lack of long-term secure funding for the development and maintenance of biobanks.  
"Although there are approximately 180 commercial biobanks in the United States with accruals of nearly 400,000 donors, no single company holds more than a 3-percent share of the global biobanking market," Vaught wrote.  
For many biobanks, he suggests, the problem of extended sustainability was at least in part due to the cyclical nature of project-specific financing that has become the norm.  
"This start-and-stop style of incrementally funding biobank projects in short durations is inconsistent with the need to annotate biospecimens with longitudinal data over an extended period of time," Vaught decried.  
Last December, Frost & Sullivan analyst Divyaa Ravishankar echoed the expansion comments of the GBI report, suggesting: "As patient population samples surge, automated storage units have now been developed that improve biobank's capacity to cater to a larger number of samples."  
Specifically, she cited advances in LIMS technologies that have led to virtual biobanks and centralized databank models that allow institutions and companies to collaborate and expand access to samples and information to researchers throughout the discovery and development chain.  
Of the 100-plus biobanks discussed in the GBI report—which covered not just stem cells, but all biological samples—68 percent were standalone facilities that received all of their funding from government, while the remainder were partnered with other biobanks or institutions.  
Increased use of automation technologies, whether in the form of liquid handling, storage management systems, consumables or LIMS, appears to help reduce some of these costs. "As cost increases, biobanks look for flexibility in their automation solutions that will connect with numerous competing instrument platforms, thus offering more testing that biobanks require in the long run," said Ravishankar in a Frost & Sullivan report published last May, which suggested that the 2011 global automation market for biobanking applications alone surpassed $800 million, and that by 2018, this market could reach $1.4 billion.  
Another way in which biorepositories can be made more cost effective is to maximize the return side of the return-on-investment equation by improving the amount and types of data generated by the biobank. According to Mark Collins, director of marketing at BioFortis, one way to do this would be to move away from a sample-centric business model and more closely link samples to the research data arising from those samples.  
"Since the research ecosystem is a dynamic, distributed system, next-generation biobanking requires a high degree of flexibility to meet the demands of a wide variety of studies occurring in the research ecosystem," Collins wrote in a recent white paper. "Indeed, the comprehensive, physical biobank with hardware, software and samples may not actually exist within this externalized, collaborative paradigm."  
Instead, Collins envisions a virtual biobanking system, in which samples may be distributed across a number of locations, each one sending samples as requested for analysis, perhaps through a contract research organization. The results of these analyses can then be made available to the entire network.   As proof of concept, Collins describes a prostate cancer biomarkers initiative at a European molecular medicine center.  
"Exploration of the data within the deep collaborative environment enabled the discovery of proteomic biomarkers of prostate cancer and led to patent filings by the organization. Furthermore, these biomarkers formed the foundation for development of new blood-based molecular diagnostics and targets for therapeutic intervention," he says.
Increased automation and informatics solutions may not be enough, however, to facilitate the changes required to keep these resources afloat and significant economies of scale may be missed because no two biobanks operate in a similar manner. According to Ravishankar, the quality, breadth and diversity of sample data collected can also vary significantly between repositories. Biorepository harmonization of samples and data streams could provide a solution to this challenge.  
"Most regional participants have modular systems and a well-established distribution chain. Global participants, providing multicomponent systems, could form strategic partnerships with these companies to leverage their distribution chain," Ravishankar says.  
The GBI report notes, however, that despite a general desire to move toward harmonization and the initial efforts of international biobanks such as the Public Population Program in Genomics (P3G), the Biobanking and Biomolecular Resources Research Infrastructure (BBMRI) and the United Kingdom DNA Banking Network (UKDNB), progress has been slow. This is not to say that all is doom and gloom in the biorepository world, though, as some groups continue to put money into the expansion of various biobanks around the world.  
In February, Lonza announced it was awarded a second contract by the U.S. National Institutes of Health (NIH) Center for Regenerative Medicine to generate iPSCs for research purposes, further expanding the NIH's collection. Last October, the organization had contracted Lonza to generate clinical-grade iPSCs under current Good Manufacturing Practices (cGMP), a critical step in getting such cells into clinical trials.
As Lonza Chief Operating Officer Stephan Kutzer explained in the announcement, he sees the latest contract as validation of the company's efforts to provide "a comprehensive service offering to support both basic and clinical iPSC research " through its Pluripotent Stem Cell Innovation Center. The agreement is for three years and could be valued up to $6.9 million.
A mere one month later, the Coriell Institute for Medical Research and Cellular Dynamics International (CDI) announced they had been awarded two multimillion-dollar grants from the California Institute for Regenerative Medicine (CIRM) to both generate a series of iPSC lines and establish a biobanking facility. In the first grant, valued at $16 million, CDI will generate three iPSC lines for each of 3,000 healthy and diseased donors, covering conditions ranging from Alzheimer's disease and autism spectrum disorders to liver and cardiovascular disease. The second $10-million grant will allow Coriell to leverage its expertise to create a biobank for those cells, in what CDI Chairman and CEO Bob Palay describes "will create the world's largest human iPSC bank."  
That's cold  
Of course, a key component to the success of any biobanking effort is the ability to freeze and thaw any cell samples while maintaining viability. This task is made much more challenging when you start working with stem cells, according to Philip Pridham-Field, market manager for biorepository products and services at AMS Biotechnology (AMSBIO).  
"What you want to do is keep your cells alive, possibly for a very long time, and ensure that they stay stem cells," he explains. "As stem cells, however, they have the potential to become any other cells, and this can happen spontaneously.  
"If you look at them funny," he adds wistfully, "they could change into liver cells or who knows what."  
Traditionally, cryopreservation has relied on combination of media and reagents such as DMSO, glycerol, oligosaccharides or antifreeze proteins, but these reagents can have a deleterious effect on cell function and viability. Thus, the search for better cryopreservatives has become a hot topic of late. As well, as stem cells move from the research bench to the clinic, there is a growing need to develop more defined solutions.  
"If you're working with stem cells toward a cellular therapy, how you grow your cells and what you use to grow them has to be considered," explains Pridham-Field. "If you're using a medium or reagents to grow your cells and make them do what you want to do and it has animal components, you need to be sure of what is in there, if there is any danger."  
For AMSBIO, the answer is StemCell Banker, a cryopreservative solution effectively made from USP-grade reagents.  
"What's in the media is proprietary, but obviously, for anyone down the line who develops a product and needs to know its precise composition, we can tell them that under a confidentiality agreement," Pridham-Field says.  
In Cell Medicine last year, Hirofumi Noguchi and colleagues published their comparison of AMSBIO's Cell Banker line of defined cryopreservative against a variety of DMSO- and glycerol-based methods on iPSCs. They found that while all of the methods preserved cells both in terms of viability and pluripotency, the best results came with the use of Cell Banker 3.  
Going large on medium  
Defined media and reagents are also becoming more relevant outside of the biorepository, as researchers find ways to reduce costs while also keeping their eye on the clinical prize. The question of cost isn't just about the consumables themselves, but also includes the labor costs associated with maintaining the cells.
"The current technology to grow human ESCs and iPSCs is very expensive," says Nicholas Asbrock, product manager of the Stem Cell/Cell Biology unit at EMD Millipore. "The current media is very expensive, and also you have to feed the cells every day."  
To address that issue, EMD Millipore introduced its new medium—PluriSTEM—at the recent International Society for Stem Cell Research (ISSCR) conference in Boston. As Asbrock explains, PluriSTEM is a small molecule-based medium that allows researchers to feed their cells every other day.  
"The media is based on a formulation by Dr. Boris Greber, which was published [in PLoS One] last year," he says. "There are three growth factors that promote the self-renewal of ESCs, but then there are two small molecules that inhibit the spontaneous differentiation: a Wnt inhibitor and a BMP inhibitor. And because EMD Millipore is associated with CalBiochem, we actually own the small molecules, so the cost can be kept very low and is very competitive with the market leader."  
An additional benefit of PluriSTEM is that it supports single-cell passaging, which can be critical when attempting to work with individual clones of cells, rather than potentially genetically heterogeneous clumps of cells.  
"With the current technology, you have to passage in clumps either manually by scoring the clump with a needle and then transfer with a pipette, or you can use enzymes that lift the colony off the substrate and then gently break the clump apart," Asbrock says. "With our media, you can use just general enzymatic reagents and plate the single cells so that the colonies grow clonally."  
As discussed earlier with cryopreservatives, there is also an industry-wide move toward more defined media, as the traditional undefined media composed of animal products tended to suffer from significant lot-to-lot variation. Because that variation alone can impact the behavior of the cells, altering passaging performance or potentially triggering unexpected differentiation, labs tend to get around the problem of media variation by lot testing to find the optimal lot for their experiments and then purchasing in bulk, wasting valuable time and resources, as well as potentially increasing waste.  
Asbrock suggests that the defined nature of PluriSTEM and its reliance on small molecules, which he argues are more consistent than proteins, should help to remove or minimize issues of lot-to-lot variation.  
There is also the question of the ultimate goal of moving stem cells to the clinic and increased pressures to grow cells in the absence of animal components from the start. The argument would suggest that by starting in defined media, the researchers wouldn't need to go back and re-evaluate different media as they moved toward the clinic.  
As David Fiorentini, vice president of scientific affairs at Biological Industries, explained so succinctly in an ISSCR presentation on clinical-grade expansion of mesenchymal stem cells (MSCs): "The more defined your system, the more reproducible your results."  
In the presentation, Fiorentini and Kansas State University's Mark Weiss described the application of Biological Industries' MSC NutriStem XF media system for cell culture expansion, attachment, dissociation and cryopreservation, which supported the long-term growth of multipotent human MSCs with potential therapeutic applications, particularly when compared to several other commercial media or Weiss's home-brewed medium. Despite the growing concern over regulatory issues, Kenneth Ludwig, business manager of Corning's Bioprocess/Life Sciences unit, suggests the move to the clinic and the question of serum versus serum-free may not be as black and white as we believe.  
"We think that serum-free is important," he relates. "However, I heard a researcher give a talk where they said they find that a little bit of serum—say, less than 1 percent—helps in the transplant phase of cell therapies. And then I heard an FDA presentation right after and they weren't so concerned about [the use of small amounts of serum]."  
Whatever the application, there was no denying that the floor of the ISSCR conference in June was littered with companies touting their latest efforts to produce fully defined, animal-free, xeno-free media or promising that such products would arrive shortly. In April, EMD Millipore announced the launch of its OsteoMAX-XF medium for the osteogenic differentiation of mesenchymal stem cells. Similarly, Brad Hamilton, director of R&D at Stemgent, talked about his company's experiences with the development of Pluriton reprogramming media developed specifically to work with Stemgent's mRNA program.
"One of the things you'll find with a lot of people is that mRNA-based reprogramming was not very reproducible in the initial publications. To fix this problem, the company developed Pluriton. It basically facilitates reprogramming, increases efficiency and decreases derivation timelines for almost system on which it's been tested," he says, including not just their mRNA reprogramming system, but also viral and other non-viral methods.  
Because Stemgent developed it from its standard hESC and iPSC medium NutriStem—which is xeno- and feeder-free—transitioning from reprogramming medium to expansion medium is seamless, according to Hamilton, thus reducing the fall-off that is often experienced in the transition.

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