"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 roomfor your stuff anymore."
With seminal technological advances such as the developmentof induced pluripotent stem cells (iPSCs), the potential for stem celltechnology has never been greater. Whether used as research tools to generatebiofactories or models of human disease (which you can read about in our August2013 issue) or as regenerative medicines or vaccine therapeutics (which you canread about in our September issue), stem cells offer researchers and cliniciansa wealth of new opportunities.
But this wealth comes with a price tag, as companies and researchorganizations try to determine how best to grow and store the myriad cell linesfor future use.
According to a report from GBI Research last July, thegrowth in biorepositories has been dramatic since the 1970s, with increases of42 percent and 36 percent in the 1990s and 2000s, respectively. Changes toresearch funding budgets, however, significantly threatens this growth at atime when demand may be at its highest.
In 2011, Jimmie Vaught and colleagues at the National CancerInstitute's (NCI) Office of Biorepositories and Biospecimen Research expressedtheir concern about the threat to future cancer research coming from the lackof long-term secure funding for the development and maintenance of biobanks.
"Although there are approximately 180 commercial biobanks inthe United States with accruals of nearly 400,000 donors, no single companyholds more than a 3-percent share of the global biobanking market," Vaughtwrote.
For many biobanks, he suggests, the problem of extendedsustainability was at least in part due to the cyclical nature ofproject-specific financing that has become the norm.
"This start-and-stop style of incrementally funding biobankprojects in short durations is inconsistent with the need to annotatebiospecimens with longitudinal data over an extended period of time," Vaughtdecried.
Last December, Frost & Sullivan analyst DivyaaRavishankar echoed the expansion comments of the GBI report, suggesting: "Aspatient population samples surge, automated storage units have now beendeveloped that improve biobank's capacity to cater to a larger number ofsamples."
Specifically, she cited advances in LIMS technologies thathave led to virtual biobanks and centralized databank models that allowinstitutions and companies to collaborate and expand access to samples andinformation to researchers throughout the discovery and development chain.
Of the 100-plus biobanks discussed in the GBI report—whichcovered not just stem cells, but all biological samples—68 percent were standalonefacilities that received all of their funding from government, while theremainder were partnered with other biobanks or institutions.
Increased use of automation technologies, whether in theform 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 theirautomation solutions that will connect with numerous competing instrumentplatforms, thus offering more testing that biobanks require in the long run,"said Ravishankar in a Frost & Sullivan report published last May, whichsuggested that the 2011 global automation market for biobanking applicationsalone surpassed $800 million, and that by 2018, this market could reach $1.4billion.
Another way in which biorepositories can be made more costeffective is to maximize the return side of the return-on-investment equationby improving the amount and types of data generated by the biobank. Accordingto Mark Collins, director of marketing at BioFortis, one way to do this wouldbe to move away from a sample-centric business model and more closely linksamples to the research data arising from those samples.
"Since the research ecosystem is a dynamic, distributedsystem, next-generation biobanking requires a high degree of flexibility tomeet the demands of a wide variety of studies occurring in the researchecosystem," Collins wrote in a recent white paper. "Indeed, the comprehensive,physical biobank with hardware, software and samples may not actually existwithin this externalized, collaborative paradigm."
Instead, Collins envisions a virtual biobanking system, inwhich samples may be distributed across a number of locations, each one sendingsamples as requested for analysis, perhaps through a contract researchorganization. The results of these analyses can then be made available to theentire network.
As proof of concept, Collins describes a prostate cancerbiomarkers initiative at a European molecular medicine center.
"Exploration of the data within the deep collaborativeenvironment enabled the discovery of proteomic biomarkers of prostate cancerand led to patent filings by the organization. Furthermore, these biomarkersformed the foundation for development of new blood-based molecular diagnosticsand targets for therapeutic intervention," he says.
Increased automation and informatics solutions may not beenough, however, to facilitate the changes required to keep these resourcesafloat and significant economies of scale may be missed because no two biobanksoperate in a similar manner. According to Ravishankar, the quality, breadth anddiversity of sample data collected can also vary significantly betweenrepositories. Biorepository harmonization of samples and data streams could providea solution to this challenge.
"Most regional participants have modular systems and awell-established distribution chain. Global participants, providingmulticomponent systems, could form strategic partnerships with these companiesto leverage their distribution chain," Ravishankar says.
The GBI report notes, however, that despite a general desireto move toward harmonization and the initial efforts of international biobankssuch as the Public Population Program in Genomics (P3G), the Biobanking andBiomolecular Resources Research Infrastructure (BBMRI) and the United KingdomDNA Banking Network (UKDNB), progress has been slow. This is not to say thatall is doom and gloom in the biorepository world, though, as some groupscontinue to put money into the expansion of various biobanks around the world.
In February, Lonza announced it was awarded a secondcontract by the U.S. National Institutes of Health (NIH) Center forRegenerative Medicine to generate iPSCs for research purposes, furtherexpanding the NIH's collection. Last October, the organization had contractedLonza to generate clinical-grade iPSCs under current Good ManufacturingPractices (cGMP), a critical step in getting such cells into clinical trials.
As Lonza Chief Operating Officer Stephan Kutzer explained inthe announcement, he sees the latest contract as validation of the company'sefforts to provide "a comprehensive service offering to support both basic andclinical iPSC research" through its Pluripotent Stem Cell Innovation Center. Theagreement is for three years and could be valued up to $6.9 million.
A mere one month later, the Coriell Institute for MedicalResearch and Cellular Dynamics International (CDI) announced they had beenawarded two multimillion-dollar grants from the California Institute forRegenerative Medicine (CIRM) to both generate a series of iPSC lines andestablish a biobanking facility. In the first grant, valued at $16 million, CDIwill generate three iPSC lines for each of 3,000 healthy and diseased donors, coveringconditions ranging from Alzheimer's disease and autism spectrum disorders toliver and cardiovascular disease. The second $10-million grant will allowCoriell to leverage its expertise to create a biobank for those cells, in whatCDI Chairman and CEO Bob Palay describes "will create the world's largest humaniPSC bank."
Of course, a key component to the success of any biobankingeffort is the ability to freeze and thaw any cell samples while maintainingviability. This task is made much more challenging when you start working withstem cells, according to Philip Pridham-Field, market manager for biorepositoryproducts and services at AMS Biotechnology (AMSBIO).
"What you want to do is keep your cells alive, possibly fora very long time, and ensure that they stay stem cells," he explains. "As stemcells, however, they have the potential to become any other cells, and this canhappen spontaneously.
"If you look at them funny," he adds wistfully, "they couldchange into liver cells or who knows what."
Traditionally, cryopreservation has relied on combination ofmedia and reagents such as DMSO, glycerol, oligosaccharides or antifreezeproteins, but these reagents can have a deleterious effect on cell function andviability. Thus, the search for better cryopreservatives has become a hot topicof 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 cellulartherapy, how you grow your cells and what you use to grow them has to beconsidered," explains Pridham-Field. "If you're using a medium or reagents togrow your cells and make them do what you want to do and it has animalcomponents, you need to be sure of what is in there, if there is any danger."
For AMSBIO, the answer is StemCell Banker, acryopreservative solution effectively made from USP-grade reagents.
"What's in the media is proprietary, but obviously, foranyone down the line who develops a product and needs to know its precisecomposition, we can tell them that under a confidentiality agreement,"Pridham-Field says.
In Cell Medicinelast year, Hirofumi Noguchi and colleagues published their comparison ofAMSBIO's Cell Banker line of defined cryopreservative against a variety ofDMSO- and glycerol-based methods on iPSCs. They found that while all of themethods preserved cells both in terms of viability and pluripotency, the bestresults came with the use of Cell Banker 3.
Going large on medium
Defined media and reagents are also becoming more relevantoutside of the biorepository, as researchers find ways to reduce costs whilealso keeping their eye on the clinical prize. The question of cost isn't justabout the consumables themselves, but also includes the labor costs associatedwith maintaining the cells.
"The current technology to grow human ESCs and iPSCs is veryexpensive," says Nicholas Asbrock, product manager of the Stem Cell/CellBiology unit at EMD Millipore. "The current media is very expensive, and alsoyou have to feed the cells every day."
To address that issue, EMD Millipore introduced its newmedium—PluriSTEM—at the recent International Society for Stem Cell Research(ISSCR) conference in Boston. As Asbrock explains, PluriSTEM is a small molecule-basedmedium 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 theself-renewal of ESCs, but then there are two small molecules that inhibit thespontaneous differentiation: a Wnt inhibitor and a BMP inhibitor. And becauseEMD Millipore is associated with CalBiochem, we actually own the smallmolecules, so the cost can be kept very low and is very competitive with themarket leader."
An additional benefit of PluriSTEM is that it supportssingle-cell passaging, which can be critical when attempting to work withindividual clones of cells, rather than potentially genetically heterogeneousclumps of cells.
"With the current technology, you have to passage in clumpseither manually by scoring the clump with a needle and then transfer with apipette, or you can use enzymes that lift the colony off the substrate and thengently break the clump apart," Asbrock says. "With our media, you can use justgeneral enzymatic reagents and plate the single cells so that the colonies growclonally."
As discussed earlier with cryopreservatives, there is alsoan industry-wide move toward more defined media, as the traditional undefinedmedia composed of animal products tended to suffer from significant lot-to-lotvariation. Because that variation alone can impact the behavior of the cells,altering passaging performance or potentially triggering unexpecteddifferentiation, labs tend to get around the problem of media variation by lottesting to find the optimal lot for their experiments and then purchasing inbulk, wasting valuable time and resources, as well as potentially increasingwaste.
Asbrock suggests that the defined nature of PluriSTEM andits reliance on small molecules, which he argues are more consistent thanproteins, should help to remove or minimize issues of lot-to-lot variation.
There is also the question of the ultimate goal of moving stemcells to the clinic and increased pressures to grow cells in the absence ofanimal components from the start. The argument would suggest that by startingin defined media, the researchers wouldn't need to go back and re-evaluatedifferent media as they moved toward the clinic.
As David Fiorentini, vice president of scientific affairs atBiological Industries, explained so succinctly in an ISSCR presentation onclinical-grade expansion of mesenchymal stem cells (MSCs): "The more definedyour system, the more reproducible your results."
In the presentation, Fiorentini and Kansas StateUniversity's Mark Weiss described the application of Biological Industries' MSCNutriStem XF media system for cell culture expansion, attachment, dissociationand cryopreservation, which supported the long-term growth of multipotent humanMSCs with potential therapeutic applications, particularly when compared toseveral other commercial media or Weiss's home-brewed medium.
Despite the growing concern over regulatory issues, KennethLudwig, business manager of Corning's Bioprocess/Life Sciences unit, suggeststhe move to the clinic and the question of serum versus serum-free may not beas 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 ofserum—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 concernedabout [the use of small amounts of serum]."
Whatever the application, there was no denying that thefloor of the ISSCR conference in June was littered with companies touting theirlatest efforts to produce fully defined, animal-free, xeno-free media orpromising that such products would arrive shortly.
In April, EMD Millipore announced the launch of itsOsteoMAX-XF medium for the osteogenic differentiation of mesenchymal stemcells. Similarly, Brad Hamilton, director of R&D at Stemgent, talked abouthis company's experiences with the development of Pluriton reprogramming mediadeveloped specifically to work with Stemgent's mRNA program.
"One of the things you'll find with a lot of people is thatmRNA-based reprogramming was not very reproducible in the initial publications.To fix this problem, the company developed Pluriton. It basically facilitatesreprogramming, increases efficiency and decreases derivation timelines foralmost system on which it's been tested," he says, including not just theirmRNA reprogramming system, but also viral and other non-viral methods.
Because Stemgent developed it from its standard hESC andiPSC medium NutriStem—which is xeno- and feeder-free—transitioning fromreprogramming 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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