(This story is the continuation/conclusion of the article beginninghere)
Keeping a full plate
According to Corning's Ludwig, another question—aside fromthe animal-free discussion—that he hears a lot is, "How can I make 2 trillioncells?"
"They never really talk about it, but there is always thisquestion of 'how can I make a trillion cells cheaply?' There is a lot ofemphasis now on cost and cost-containment," he says.
This question will only get louder as more and more projectsmove from the research departments and into the clinic, and the demands forlarge quantities of cells increases. With standard polystyrene plates, Ludwigexplains, you can really only get stacks of about 40 plates because of the needfor sufficient headspace between the plates to allow adequate gas exchange.
"If you reduce the headspace, you get poor oxygen transferand the cells end up starving," he says.
To accommodate the demand for tighter stacking and increasedthroughput, Corning introduced the HyperStack format, which relies on agas-permeable polystyrene. Thus, oxygen transfer occurs through the permeablefilm allowing the company to create a very dense stack.
"But even with something like HyperStack, you can reallyonly get about 2 billion cells with a 120-layer stack, so customers are reallyactively looking at bioreactors with microcarrier beads," he adds.
"As more cell-based therapeutics progress toward clinicaltesting, the consistency, quality and reproducibility of large-scale culturesystems become an imperative," said Robert Shaw, commercial director of EMDMillipore's Stem Cell Initiative, in a May press release.
EMD Millipore was announcing its collaboration withPharmaCell BV to optimize large-scale expansion and harvesting of HepaRG cellsusing Mobius CellReady disposable bioreactor technology. In support of theEuropean BALANCE project, the collaboration is aimed at the development of abioartificial liver.
"Customers tend to like the flexible manufacturing formatthat a single-use bioreactor gives them, so they can quickly change overwithout doing steam-in-place processes," Ludwig says. "The beads we have are alittle more differentiated—because Corning is really big on surface science—sothe advantage that we have in the stem cell area is that we have surfacetreatments, media and the vessels together, which makes it easier for thecustomers looking for a transitional system."
Several posters at the ISSCR conference were dedicated tothe development of microcarrier technologies, and the challenges of shiftingfrom a two-dimensional platform to the third dimension of suspended cultures.
"The biggest thing around the microcarrier beads is thatthey seed differently; you can't just put some beads in suspension with somecells, you have to let them settle," Ludwig adds. "Developing that kind ofprotocol to get them to seed on the beads and expand correctly is a little bitchallenging."
As he explains further, it is not just about the surfacematerials per se and their celladhesion properties.
"It's a much more dynamic environment," he says. "Wheneveryou have things in motion, there are lots of concerns about how themicrocarriers are interacting with the impellers, and making sure that theoxygen level and heat is maintained from top to bottom."
A transitional offering between plates and a full bioreactorenvironment may be in the offing, however, with the announcement in April ofdata arising from a collaboration between UCB and TAP Biosystems. Inparticular, UCB tested the performance of a variety of cell lines in the 15-mLambr microscale bioreactor and found the results were representative of similarexperiments with 100 L bioreactors, and yet would allow researchers to test upto 24 clones in parallel.
While the experiments were performed on protein-expressingCHO cell lines rather than stem cells, the results nonetheless open thepossibility of testing performance at a smaller, more experimental level beforescaling up to production levels.
Aside from the growth vessels, the surface chemistry is alsoimportant to stem cell research, and this is one area where AMSBIO has investedheavily.
"Unlike cancer cells, stem cells don't tend to grow onplastic happily, but require some kind of matrices," says Pridham-Field.
Thus, AMSBIO has developed an extensive portfolio ofextracellular matrices (ECMs), but whereas many companies simply offer the ECMproteins such as laminins, AMSBIO has gone one step further.
Rather than just extracting the protein for a recombinantlaminin from a cell line, the company has developed recombinant versions wherethe cell-binding motifs of the ECM protein are fused to a naturally adhesiveprotein derived from mussels that makes it easier to stick the ECMs to plates.
"You can coat your plate with just one of these motifs, orany combination of these motifs in any concentration," says Pridham-Field. "Theidea is to grow your stem cells on these defined matrices and optimize theenvironment."
One of the challenges for researchers, he adds, is knowingwhich motifs to use for their specific experiments, a question that AMSBIO doesnot really have the capacity to answer.
"Because we recognize there is a limit as to how much we cantell people, we'll put together a 96-well plate of various motif combinations,"he says, describing something akin to a chocolate sampler box. "They can thenput the same cells in each well and see how they react."
As a follow-through on its purchase of Discovery Labware,Corning too continues to develop a portfolio of peptide surface treatments.
"Discovery Labware had invested heavily in peptide surfacetreatments, so it really complimented Corning quite well," recounts Ludwig, butwhere Corning had focused its efforts on letting the customer coat the plates,Discovery Labware focused on precoated cultureware, a practice that Corning haslargely adopted.
The battle continues to expand the repertoire of currentstem cell applications, while keeping an eye on the downstream possibilities ofsending those same cells into the clinic as therapeutics—which means that asresearchers and companies do their best to figure out how to make stem cellresources less expensive and more effective, others will continue to search forplaces to put all this stuff.
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