Growing demand of stem cells in regenerative medicine led scientists to expedite the production of these cells. Researchers at the A*STAR Bioprocessing Technology Institute (BTI) in Singapore have designed a process for mass-production of stem cells. Dr Steve Oh’s group has been growing stem cells by conventional means at BTI for seven long years, when in 2008 his colleague Shaul Reuveny proposed an idea for mass-production.
Stem cells are normally cultured in Petri dishes. Instead of culturing the cells on round, flat Petri dishes, he could try growing them on tiny polystyrene beads known as microcarriers floating in a nutritional brew, suggested by Reuveny, a visiting scientist at BTI now. This technique had been used for decades to mass-produce virus-infected cells for the vaccine industry, which Reuveny had expertise in this area.
The average Petri dish fits fewer than 100,000 cells, a miniscule amount when stacked against the 2 billion muscle cells that make up the heart or the 100 billion red blood cells needed to fill a bag of blood. The approach Reuveny suggested potentially could produce cells in much vaster numbers to make them more practical for therapy.
“Why don’t I bring some of these microcarriers over to you?” Reuveny suggested to Oh. Eventually, Oh was convinced to try what could become the most scalable method for growing stem cells and differentiated cells worldwide.
“There is a trend now in industry to move away from this simple Petri-dish method to manufacturing stem cells in bioreactor processors,” Oh says. “We started this journey much earlier than everyone else in the world.”
Oh’s Stem Cell Group first tried the approach on human embryonic stem cells. These are found in the early embryo and have the potential to mature into any type of cell in the body, a state known as pluripotency. For months, they struggled to develop a coating that would make the stem cells stick to the microcarriers, and to formulate a solution that contained the right mixture of nutrients for the cells to grow. “Without Reuveny’s know-how, we probably would have failed,” says Oh.
About a year into their hard work, one line of human embryonic stem cells survived past the 20-week mark of stability. Not only were these cells viable, they were also two to four times more densely packed than those grown in Normal Petri dishes.
The group has spent the last six years refining their processes to produce even more cells using cheaper materials and fewer steps. “We are easily achieving three times higher cell densities than the Petri dish approach,” says Oh. “In some cases, by modifying the feeding strategy, we can get six times more cell densities, and we could probably reach ten with a bit of work.”
The process can be scaled up exponentially in larger tanks. “If in one week we can go from a ten-milliliter (10mL) culture volume to a hundred-milliliter (100mL) bioreactor, then the next week we can go from a hundred milliliters to one liter; and ten liters the week after that,” explains Oh. The equivalent in Petri dishes from 100 to 1,000 to 10,000 would be practically impossible for a researcher to handle.
The team has also expanded their repertoire to two other types of stem cells induced pluripotent stem cells and adult mesenchymal stem cells as well as differentiated heart, neural, bone and red blood cells.
Therapeutic strategy for hearts
The biggest advances for Oh’s team in recent years have been in the growth of differentiated heart muscle cells, called cardiomyocytes. “We beat the Petri dish method on all counts—purity, yield, cost of goods and simplicity of process,” he maintains.
But a lot of their success is thanks to protocols initially developed on Petri dishes. For a start, cardiomyocytes are the fastest cell type to differentiate, taking only two weeks. And researchers have developed a method to grow pure batches of cardiomyocytes without the addition of expensive growth factors. Instead, they use small molecules to first inhibit and then activate a key cell-differentiation pathway known as Wnt signaling. Oh’s team applied this small-molecule approach to grow and differentiate cardiomyocytes from embryonic stem cells directly on their microcarriers.
The ultimate goal of the research is to grow enough cells in an affordable way to patch up one square-centimeter of damaged heart muscle following a heart attack.
Oh’s team is now partnering with industry to further improve the process, as well as with clinicians to test the healing potential of their cells on animal models. “We always try to keep the end in mind, to translate our work into something that can eventually be used by companies and clinicians.”
Heart cells are just one cell type being grown at BTI. From embryonic stem cells, the team has also developed progenitor cells halfway to becoming mature neurons, as well as dopaminergic neurons that when progressively lost in the brain can cause Parkinson’s disease. And the team is in the early stages of differentiating red blood cells at speeds and scales sufficient for use in emergency blood transfusions.
From mesenchymal cells, for which there are currently more than 400 registered clinical trials worldwide, Oh’s group is growing bone and cartilage cells known as osteoblasts and chondrocytes that can be introduced to animal models to repair damaged tissue.
Preliminary results on the ability of scalably grown stem cells to differentiate into osteoblasts and produce cell-signaling cytokine molecules have been encouraging.