If you freeze one million embryonic stem cells, less than one percent will survive, say Professors Juan de Pablo and Sean Palecek. Consequently, in storing stem cells for future experiments, scientists must freeze many millions in order to have enough to culture once thawed. Embryonic stem cells are of intense interest to medicine and science because of their ability to develop into virtually any other cell made by the human body. In theory, if stem cells can be grown and their development directed in culture, it would be possible to grow bone marrow, neural tissue, muscle or any of the other 220 cell types that make up the tissues and organs in the body.
A complete understanding of what happens to stem cells when frozen will not only shed light on how these amazing cells function but also could lead to a day when freeze-dried stem cells are stored at room temperature, shipped around the world and rehydrated to heal injuries. Working with stem cell pioneer James Thompson and the WiCell Research Institute, de Pablo and Palecek. are conducting a physical, genomic and biochemical analysis of where damage occurs to platelets and stem cells during freezing and freeze drying, as well as the factors that help them survive. The project is part of a three-year, $1.3 million Defense Advanced Research Projects Agency (DARPA) grant.
“An important component of our efforts is devoted to the freeze-drying and storing of platelets,” says de Pablo. “Platelets are the component of your blood necessary for wound healing. So when there is an accident or civil disaster platelets are in high demand; because these cells facilitate the healing process. With platelets, a patient can hopefully survive until you find the necessary supply of blood. Currently there is no product to supplement the loss of platelets. But we’ve been working on this and have been fairly successful. In the future it would be tremendous to apply similar techniques to red blood cells. If successful, it would change the way blood is collected and stored, so we are excited about that.”
But the researchers say stem cells are much more delicate and complicated. Their viability after freezing is worse than just about any other cell type known. The process causes a wide range of both physical and chemical damage as well as biological harm. For example, ice crystal formation can break cell membranes or destroy fragile cell structures like microtubules that separate chromosomes. Those issues might be addressed by making chemical changes to the cryopreservation solution or by changing the freezing rate; Faster freezing results in smaller ice crystals. Biological damage presents still more challenges. “We really don’t understand the damage on the biological side very well, but we know that when you try to preserve the cells, they begin to differentiate,” Palecek says.
“So they lose the embryonic stem cell character and we’re not sure why that happens. We can measure that they do differentiate but we’re not sure what the mechanism is.”
It could be that chemical protectants, stresses of temperature change, or changes in osmolarity of the solution are triggering the cells to differentiate. The team’s approach is to look at the factors known to cause cell death or differentiation through chemical signaling and try to maintain those factors during the freezing process while changing others. For example, the solvent DMSO (dimethylsulfoxide) is a membrane permeable protectant commonly used in cell preservation, but it is particularly toxic to embryonic stem cells. If you don’t use any, Palecek says, all of the cells die.
“So you kill a large number of them so you can save a small number of them,” he says. “We’re looking at a number of things that are a bit more cell friendly-sugars for example. The problem with sugars is that you can’t get them into the cell at nearly the same level because they are not permeable to the cell membrane. So we’re trying to figure out ways to load these sugars into the cell and stabilize the cell that way.” Additionally, the team is analyzing gene expression patterns before and after freezing to determine the cells’ temporary response and longer term or permanent response to the process. Cells respond to stress by increasing expression of certain genes while decreasing expression of others. Understanding when and what the cell produces could offer strategies to help the cell survive.
“We take a multipronged approach,” says de Pablo. “We do theory. We do calculations that tell us how a cell membrane might respond. We use very careful characterization of cells as we freeze and dry them. We use calorimetry, microscopy and X-rays, and we conduct gene analysis. And by putting all of this data together we hope to develop a picture of what exactly happens to a cell when we freeze it and dry it.” The science of stem cells is still in its infancy. While clearly important, there is no way to predict the ultimate demand for the cells. The researchers say there is a big difference in creating procedures for preserving million of cells compared to preserving billions of cells. But in the next couple of years the team hopes to have new methods that are orders of magnitude better in viability and preventing differentiation than the current standard.
“The current standard is based on what worked for other cell types,” says Palecek. “That’s clearly not good enough.”