With a Kobe University-developed procedure, induced pluripotent stem cells can now be frozen directly in their dishes without losing their viability or undifferentiated state after thawing. This marks a significant step for research automation, personalized medicine and drug discovery research.
Induced pluripotent stem cells, also widely known as iPS cells, can be created from any tissue in the human body and possess the ability to transform into a wide range of tissues. As such, they are essential for regenerative medicine and drug discovery research. Kobe University biochemical engineer MARUYAMA Tatsuo says, "However, their production, handling and cryopreserved storage requires advanced technology and skilled staff, making their use very expensive and not suited for automation or mass production." One critical bottleneck so far has been that there is no technique for cryopreserving the cells directly in the culture dish they are grown in as two-dimensional (2D) sheet cultures.
"We previously investigated the cryopreservation of 2D cancer cells cultures and discovered that the amino acid 'D-proline' showed remarkable efficacy as a cryoprotectant. When we talked about this with a researcher at the medical school, we learned that if we could apply this to cryopreserving iPS cells, this would be useful for regenerative medicine," explains Maruyama. Moreover, while his group previously confirmed that animal-derived components are effective cryoprotectants for mammalian cells, they are not suitable for stem cell cultures. This is because such animal-derived substances may induce the stem cells to differentiate, meaning they lose their pluripotency, and pose a risk of contamination for downstream applications.
In the Biochemical Engineering Journal, Maruyama and his team now published that they found a cryopreservation method for iPS cell sheet cultures. Importantly, they showed that their method almost perfectly preserves the cells' viability even after three months of freeze storage while also maintaining their pluripotency. The study's first author MORITA Kenta says: "We succeeded using D-proline, an inexpensive amino acid, as the primary cryoprotectant. We showed that it is just as effective for traditional, suspended iPS cell cultures as conventional cryoprotectants, but we are the first to publish a solution for 2D sheet cultures in their culture dishes."
A key step in their protocol was that, prior to freezing, they weakened cell-cell adhesion through a brief enzymatic reaction. This not only allowed the cryoprotectant to penetrate the cells more easily but also reduced physical stress onto the cells, reducing freezing damage. "The method we developed here is straightforward and will make it easy to automate the cryopreservation of iPS cells," Morita explains.
iPS cells can be used to create heart, nerve, blood, muscle and other tissues for studying drug efficacy, for replacement of lost or damaged tissue, and for other future therapies. In light of this, group leader Maruyama says: "If our technology enables the direct cryopreservation of iPS cell sheets, maintaining them will become much easier than it is today. Automating cryopreservation and thawing using robots, along with the ability to use them immediately after thawing for research or treatment, will accelerate personalized medicine for individual patients and drug discovery research."
This research was funded by the Nakatani Foundation for Advancement of Measuring Technologies in Biomedical Engineering (grant 2022S230), the Toyota Physical and Chemical Research Institute, the NORITZ Nukumori Foundation (grant RS2408), the Takeda Science Foundation, Koyanagi Zaidan, the Canon Foundation, and the Japan Society for the Promotion of Science (grants 19H05458, 23H01774, 23K13610, Program for Forming Japan's Peak Research Universities (J-PEAKS)). It was conducted in collaboration with a researcher from the Osaka Metropolitan University.
Kobe University is a national university with roots dating back to the Kobe Higher Commercial School founded in 1902. It is now one of Japan's leading comprehensive research universities with over 16,000 students and over 1,700 faculty in 11 faculties and schools and 15 graduate schools. Combining the social and natural sciences to cultivate leaders with an interdisciplinary perspective, Kobe University creates knowledge and fosters innovation to address society's challenges.
