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Researchers at Kobe University, led by MARUYAMA Tatsuo, have developed a novel method to freeze induced pluripotent stem cell (iPS cell) sheets—an advancement that preserves their quality during long-term storage.

Credit: UCHIDA Satoru

This new approach enables iPS cells to be cryopreserved directly within their culture dishes while maintaining their vitality and undifferentiated state after thawing. It represents a key advancement in streamlining research processes and enhancing applications in personalized medicine and pharmaceutical development.

iPS cells are derived from various tissues in the human body and can develop into numerous other cell types. Their versatility makes them essential in areas like tissue regeneration and medical testing. However, their production and storage typically require specialized technology and skilled expertise, which limits scalable use. One enduring challenge has been finding a convenient method to freeze these cells in their two-dimensional sheet culture forms without transferring them to new containers.

“Our previous work on freezing 2D cancer cell cultures showed that the amino acid D-proline helped protect cells during freezing,” notes Maruyama. “Speaking with colleagues in medical science, it became clear that applying this method to iPS cells could significantly aid regenerative research.” However, while animal-derived cryoprotectants were effective in earlier studies, they are unsuitable for iPS cells due to the risk of contamination and unintended cell differentiation.

Publishing their findings in the Biochemical Engineering Journal, Maruyama and his group demonstrated a reliable preservation technique for iPS cell sheets. Significantly, the technique retained cell health and maintained their stem-cell properties even after being stored frozen for three months. MORITA Kenta, the study’s lead author, explained: “We succeeded in using D-proline—a cost-effective amino acid—as the main cryoprotectant. While it works comparably well with traditional cell suspensions, our method is the first to offer a practical solution for 2D sheet cultures within their original dishes.”

A defining step in their procedure involved a quick enzymatic reaction before freezing that weakens the bonds between cells. This not only makes it easier for the protective agent to reach the cells but also lowers the risk of damage from freezing. “Our method is simple and suitable for automation,” Morita added.

iPS cells can potentially become heart, nerve, blood, and muscle tissue, making them vital for therapies and testing new treatments. Maruyama emphasized the broader impact: “With the ability to freeze and preserve iPS cell sheets directly in their culture dishes, maintenance becomes more manageable. The integration of robotic systems for freezing and thawing makes it possible to use these cells immediately, paving the way for more personalized treatment options and accelerating drug research.”

This study was supported by funding from the Nakatani Foundation for Advancement of Measuring Technologies in Biomedical Engineering, the Toyota Physical and Chemical Research Institute, the NORITZ Nukumori Foundation, the Takeda Science Foundation, Koyanagi Zaidan, the Canon Foundation, and the Japan Society for the Promotion of Science. The work was carried out with collaboration from Osaka Metropolitan University.

Kobe University, established in 1902 as Kobe Higher Commercial School, is a leading national academic institution in Japan. With more than 16,000 students and over 1,700 faculty members across 11 faculties and 15 graduate schools, the university emphasizes interdisciplinary education that addresses complex societal challenges.

Journal

Biochemical Engineering Journal

DOI

10.1016/j.bej.2025.110041

Method of Research

Experimental study

Subject of Research

Human tissue samples

Article Title

Ready-to-use cryopreservation of undifferentiated induced pluripotent stem cells (iPSCs) without detachment from culture plates using D-proline and a synthetic polymer

Article Publication Date

6-Dec-2025