Kobe University develops new cryopreservation method to preserve iPS cells as intact sheets, advancing personalized medicine

A research group led by Assistant Professor Kenta Morita, Professor Tatsuo Maruyama, and Researcher Tomoko Yashiro from the Graduate School of Engineering, together with Professor Takashi Aoi and his colleagues from the Graduate School of Medicine at Kobe University, has successfully developed a preservation solution which enables the undisrupted cryopreservation of human iPS cells cultured in sheet form. Applications of this achievement in regenerative medicine and diagnostic systems are anticipated in the future. Their findings were published in Biochemical Engineering Journal.

iPS cells are essential for regenerative medicine and drug discovery research, but their handling demands advanced techniques and high costs. Conventional cryopreservation of iPS cells has required multiple steps: (1) detaching cells from the culture vessel and dissociating them into individual cells, (2) collecting the cells using centrifugal force, and (3) re-dispersing them in preservation solution before freezing. This process has been unsuitable for automation or mass production. Notably, no technology had been established for cryopreserving cells while they remain attached to the culture vessel (adherent culture).

The newly developed cryopreservation solution is composed of dimethyl sulfoxide (a classic cryoprotectant), cell culture medium, water, D-proline, a synthetic polymer (PDEGMA-b-PMPC-b-PDEGMA), and sodium chloride.

D-proline is an amino acid with enzyme-stabilizing properties that enhances the freezing tolerance of iPS cells. The synthetic polymer functions to prevent large ice crystals from growing inside cells. A combination of D-proline and the synthetic polymer demonstrated high cryopreservation effectiveness for iPS cells in adherent culture.

Prior to being frozen, the cells were briefly treated with an enzyme that weakens cell adhesion. Through this, the researchers successfully loosened the bonds between cells without destroying the cell sheet. Microscopic observation confirmed that the cell sheet was maintained even though the bonds between cells were weakened. This allowed the preservation solution to penetrate the cells more easily, thus significantly reducing potential freezing damage. On the other hand, when the same method was attempted using commercially available cryopreservation solutions for iPS cells, adherent iPS cells could not be successfully cryopreserved. The effect is only achieved when the enzyme pretreatment is combined with the cryopreservation solution developed by the research group.

If this technology enables the cryopreservation of iPS cell sheets as intact sheets, the maintenance and management of iPS cells will become easier than before. The cryopreservation and thawing of iPS cells could be automated using robots, and the cells could be used immediately after thawing for research or treatment. This would enable faster progress in personalized medicine for individual patients as well as accelerated drug discovery research.

Journal Information
Publication: Biochemical Engineering Journal
Title: Ready-to-use cryopreservation of undifferentiated induced pluripotent stem cells (iPSCs) without detachment from culture plates using D-proline and a synthetic polymer
DOI: 10.1016/j.bej.2025.110041