Of the many characters Australian actor Hugh Jackman has played, Marvel Comics’ Wolverine is arguably his most iconic role.
Underlying Wolverine’s longevity in the often violent and cataclysmic comic-book universe is his powerful regenerative ability, conferred by something vaguely referred to as a ‘healing factor.’ Although greatly exaggerated for dramatic effect in the comics, ‘healing factors’ do exist in the human body and can take the form of stem cells.
In adults, stem cells can be found in a variety of organs, including bone marrow, liver, skin and skeletal muscle. Unlike mature cells that are specialized and that do not divide, adult stem cells retain their ability to self-renew and are pluripotent, which means that their fates and functions are not yet cast in stone. For example, mesenchymal stem cells (MSCs)—a type of stem cell typically found in umbilical cord blood and bone marrow—can give rise to bone, brain, fat and muscle cells.
When thought of as replacement parts for machines, stem cells represent a novel class of treatments that could help repair organs damaged by injury or disease. However, hurdles remain in bringing these living medicines to the clinic.
“As the cell therapy market expands and more cell therapies get approved, there is a need to be able to manufacture cells at a large scale in order to deliver these therapies to patients on time,” May Win Naing, Head of the Bio-Manufacturing Programme at A*STAR’s Singapore Institute of Manufacturing Technology (SIMTech), told A*STAR Research.
Some cell therapies that have received regulatory approval include TEMCELL® for the treatment of acute Graft versus Host disease in Japan, and Alofisel® for the treatment of Crohn’s disease in Europe. Meanwhile, hundreds of other clinical trials involving stem cells are currently underway.
“Manufacturing cells for these applications must be carried out with safety, consistency, scalability, reproducibility and comparability in mind,” Win Naing added.
A numbers game
Scientists estimate that 1–10 million MSCs per kilogram of patient are required for each infusion of a cell-based therapy. The massive number of cells needed for multiple infusions (further multiplied by many patients) means that traditional cell culture methods fall short, primarily because of limitations in the surface area available for cell growth, as well as difficulties in maintaining a homogenous physical and chemical environment for the cells.
Seeking to overcome these challenges, researchers led by Steve Oh, Director of the Stem Cell Bioprocessing group at A*STAR’s Bioprocessing Technology Institute (BTI), developed biodegradable microcarriers that can be used to expand MSC populations in vitro. The research also involved collaborators at the Institute of Materials Research and Engineering (IMRE) and the Singapore Bioimaging Consortium (SBIC).
Fundamentally, microcarriers are tiny, porous particles with a large surface area to volume ratio for MSC attachment and growth. “Our microcarriers are made of polycaprolactone—a cheap, consistent material source widely used as a biodegradable construct for scaffolds and reconstruction of bone,” said Oh, adding that his team coated the microcarriers in a layer of three proteins that promote MSC adhesion and spreading.
With a density of just 1.06 g/cm3 , these coated microcarriers were easily suspended under constant stirring in well-defined culture media—liquid containing a precise combination of nutrients and other chemicals that encourage MSC growth while preventing MSCs from losing their pluripotency. Using their system, the researchers achieved cell densities of nearly half a million cells per milliliter of cell culture—about a ten-fold increase over conventional culture techniques. More than 90 percent of those cells were viable and expressed markers typical of MSCs.
Further, when applied to a defect inflicted on the calvarial bone, or skullcap, of rats, MSCs delivered with the coated microcarriers enhanced bone healing as compared to MSCs or microcarriers alone. This result suggests that the coated microcarriers serve as a support matrix, acting synergistically with the MSCs to regenerate bone. Another crucial observation was the absence of inflammatory cells at the site of MSC-on-microcarrier delivery, indicating that there was no rejection of the graft.
Having demonstrated the feasibility of high-density MSC culture using coated microcarriers and proved their therapeutic efficacy in rats, the researchers are now looking to scale up their approach. “We will be working with IMRE to mass produce the biodegradable microcarriers to kilogram levels for ten-liter scale bioprocessing,” Oh said. The ultimate goal, he added, is to create a bioreactor capable of producing billions of MSCs of consistent quality for use in the clinic.
Separating the good from the bad
But microcarrier properties are not the only considerations when building a good bioreactor, said Oh. “Mixing becomes an issue if the microcarrier concentration is increased, and this puts an upper limit on cell densities in culture,” he explained. “Furthermore, cell expansion in bioreactors is inevitably accompanied by some unwanted byproducts such as particulate contamination, including dead cells and microcarrier debris.”
To develop methods for sifting out unwanted contaminants in bioreactors without compromising the quantity and quality of the cell-bearing microcarriers, Oh collaborated with Win Naing’s team at SIMTech. Instead of using a membrane as a filter, the scientists designed a spiral microfluidic system that relies on flow forces within liquids as a sorting mechanism. The research also involved Chun Yang, Professor at Nanyang Technological University, Singapore, as well as an industry partner, Whirlcell Technologies.
Existing microfluidic systems for processing biological samples typically deal with particles about ten micrometers in size. However, microcarriers—especially those with cells attached, or those that clump—are at least ten times larger. The researchers thus had to increase the dimensions of their device and optimize its flow-control properties, depending on numerical simulations to facilitate their design. The outcome was a trapezoidal spiral channel with an outer wall that was taller than the inner wall.
Applying their scaled-up microfluidic system to a prototype bioreactor for MSCs, Win Naing’s team was able to replace ‘old’ or conditioned cell culture medium with fresh medium via a single-loop trapezoidal spiral channel. They reported 1–2 percent losses of MSC-bearing microcarriers during the medium replacement process, but showed eight-fold cell expansion over the culture period, a result comparable to standard spinner flask cultures. Notably, the size of microcarrier clumps in their prototype bioreactor was smaller, which makes for easier separation and harvesting of MSCs later on.
“Our scaled-up inertial microfluidic device relies only on hydrodynamic forces—shear force and centrifugal force—in curved channels to separate culture medium from microcarriers. Simultaneously, the isolated microcarrier-cell complexes are recycled to the bioreactor continuously, without any interruption to the operation of the bioreactor,” said Win Naing. The technique developed in this study could be applicable to the mass-culturing of immune cells for immunotherapy, or even lab-grown meat, she added.
Niches but not silos
Recognizing that their research projects are links in a broader manufacturing value chain for cell-based therapy, both Oh and Win Naing see great benefit in collaborating and combining the strengths of their respective labs.
“The Bio-Manufacturing Programme at SIMTech works closely with collaborators such as BTI and the National Cancer Center Singapore. We pursue feedback and conduct extensive validation studies to benchmark innovative manufacturing platforms and technologies for scaling up and scaling out cell therapies,” Win Naing commented. “Our capabilities at SIMTech include design and simulation software, prototyping equipment, automation and bonding technologies.”
All this is complemented by BTI’s focus on the bioprocessing aspects of cell therapies. The key thrusts at BTI revolve around the development of serum-free media for microcarrier cultures and more optimal feeding strategies to achieve higher yields and better harvesting conditions. David Fiorentini, Vice President for Scientific Affairs at Biological Industries, a multinational biotechnology company headquartered in Israel, notes that these objectives align well with industry needs.
“We entered into a collaboration with A*STAR’s BTI three years ago to develop culture medium, auxiliary solutions and processes for three-dimensional culture of MSCs in a microcarrier suspension culture system. The development work, which is still ongoing at BTI, will support the requirements for a high number of qualityassured cells to be used in clinical applications,” he said.
Meanwhile, the Bio-Manufacturing Programme at SIMTech has also inked collaborations with local manufacturers Cal-Comp Precision and MClean Technologies to expand its technical capabilities at the intersection of engineering and biology, said Win Naing.
So just as Wolverine didn’t succeed on his own but was aided by the diverse strengths of his fellow X-Men, a multidisciplinary approach bridging academia and industry is the preferred strategy for unlocking the potential of cell therapies.
“Close collaboration between clinicians, regulators, engineers and scientists—that’s what is needed to ensure cell therapies are successful,” Oh concluded.
The A*STAR-affiliated researchers contributing to this research are from the Bioprocessing Technology Institute (BTI), the Institute of Materials Research and Engineering (IMRE), the Singapore Bioimaging Consortium (SBIC) and the Singapore Institute of Manufacturing Technology (SIMTech).