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A place for my cells (continued)
(This story is the continuation/conclusion of the article beginning here)
Keeping a full plate
According to Corning's Ludwig, another question—aside from the animal-free discussion—that he hears a lot is, "How can I make 2 trillion cells?"
"They never really talk about it, but there is always this question of 'how can I make a trillion cells cheaply?' There is a lot of emphasis now on cost and cost-containment," he says.
This question will only get louder as more and more projects move from the research departments and into the clinic, and the demands for large quantities of cells increases. With standard polystyrene plates, Ludwig explains, you can really only get stacks of about 40 plates because of the need for sufficient headspace between the plates to allow adequate gas exchange.
"If you reduce the headspace, you get poor oxygen transfer and the cells end up starving," he says.
To accommodate the demand for tighter stacking and increased throughput, Corning introduced the HyperStack format, which relies on a gas-permeable polystyrene. Thus, oxygen transfer occurs through the permeable film allowing the company to create a very dense stack.
"But even with something like HyperStack, you can really only get about 2 billion cells with a 120-layer stack, so customers are really actively looking at bioreactors with microcarrier beads," he adds.
"As more cell-based therapeutics progress toward clinical testing, the consistency, quality and reproducibility of large- scale culture systems become an imperative," said Robert Shaw, commercial director of EMD Millipore's Stem Cell Initiative, in a May press release.
EMD Millipore was announcing its collaboration with PharmaCell BV to optimize large-scale expansion and harvesting of HepaRG cells using Mobius CellReady disposable bioreactor technology. In support of the European BALANCE project, the collaboration is aimed at the development of a bioartificial liver.
"Customers tend to like the flexible manufacturing format that a single- use bioreactor gives them, so they can quickly change over without doing steam-in-place processes," Ludwig says. "The beads we have are a little more differentiated—because Corning is really big on surface science—so the advantage that we have in the stem cell area is that we have surface treatments, media and the vessels together, which makes it easier for the customers looking for a transitional system."
Several posters at the ISSCR conference were dedicated to the development of microcarrier technologies, and the challenges of shifting from a two-dimensional platform to the third dimension of suspended cultures.
"The biggest thing around the microcarrier beads is that they seed differently; you can't just put some beads in suspension with some cells, you have to let them settle," Ludwig adds. "Developing that kind of protocol to get them to seed on the beads and expand correctly is a little bit challenging."
As he explains further, it is not just about the surface materials per se and their cell adhesion properties.
"It's a much more dynamic environment," he says. "Whenever you have things in motion, there are lots of concerns about how the microcarriers are interacting with the impellers, and making sure that the oxygen level and heat is maintained from top to bottom."
A transitional offering between plates and a full bioreactor environment may be in the offing, however, with the announcement in April of data arising from a collaboration between UCB and TAP Biosystems. In particular, UCB tested the performance of a variety of cell lines in the 15-mL ambr microscale bioreactor and found the results were representative of similar experiments with 100 L bioreactors, and yet would allow researchers to test up to 24 clones in parallel.
While the experiments were performed on protein-expressing CHO cell lines rather than stem cells, the results nonetheless open the possibility of testing performance at a smaller, more experimental level before scaling up to production levels.
Aside from the growth vessels, the surface chemistry is also important to stem cell research, and this is one area where AMSBIO has invested heavily.
"Unlike cancer cells, stem cells don't tend to grow on plastic happily, but require some kind of matrices," says Pridham-Field.
Thus, AMSBIO has developed an extensive portfolio of extracellular matrices (ECMs), but whereas many companies simply offer the ECM proteins such as laminins, AMSBIO has gone one step further.
Rather than just extracting the protein for a recombinant laminin from a cell line, the company has developed recombinant versions where the cell-binding motifs of the ECM protein are fused to a naturally adhesive protein derived from mussels that makes it easier to stick the ECMs to plates.
"You can coat your plate with just one of these motifs, or any combination of these motifs in any concentration," says Pridham-Field. "The idea is to grow your stem cells on these defined matrices and optimize the environment."
One of the challenges for researchers, he adds, is knowing which motifs to use for their specific experiments, a question that AMSBIO does not really have the capacity to answer.
"Because we recognize there is a limit as to how much we can tell 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 then put 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 surface treatments, so it really complimented Corning quite well," recounts Ludwig, but where Corning had focused its efforts on letting the customer coat the plates, Discovery Labware focused on precoated cultureware, a practice that Corning has largely adopted.
The battle continues to expand the repertoire of current stem cell applications, while keeping an eye on the downstream possibilities of sending those same cells into the clinic as therapeutics—which means that as researchers and companies do their best to figure out how to make stem cell resources less expensive and more effective, others will continue to search for places to put all this stuff.