Confocal microscopy images showing live/dead staining of 3D cell constructs. The top row shows a fresh spheroid. The middle row shows the outcome of conventional cryopreservation and the bottom row the outcome of the new approach, which yields a much greater fraction of live cells post-thaw. Credit: University of Warwick
Three-dimensional cell models that mimic the structure and function of organs are improving biomedical research and reducing reliance on animal models. However, one of the challenges of working with 3D cellular assemblies is the risk of damaging intracellular ice formation during preservation, due to cryoprotectants like dimethyl sulfoxide (DMSO) not fully permeating through the 3D structure. Researchers at the University of Warwick have proposed a new method for improving the cryopreservation of complex cell models, utilizing ice nucleating molecules from nature to prevent potentially deadly intracellular ice formation.
The new storage method involves including sterilized pollen washing water (PWW) along with DMSO in cryopreservation in order to leverage the soluble ice nucleating polysaccharides found in the pollen. The PWW was prepared from Carpinus betulus, a birch tree known as the European hornbeam, and added to cells in 96-well plates with 10% DMSO. The researchers first used cryomicroscopy to observe ice formation in cell monolayers cooled to -80°C with or without PWW and found that up to 50% of cells without PWW showed intracellular ice formation, while less than 10% of cells preserved with PWW had intracellular ice formation. The PWW prevents ice crystals from forming intracellularly because ice nucleators in the pollen promote ice crystal formation outside the cells at a relatively high temperature of about -8°C. Extracellular ice formation is preferable to, and helps prevent, intracellular ice formation, and this method does not require full permeation throughout the cell assembly to have a protective effect.
The researchers then tested their method using 3D cellular spheroids, similarly cooling them to -80°C in 96-well plates with 10% DMSO and with or without PWW treatment. Twenty-four hours after thawing, the team used an ATP content assay to assess viability of the cells after cryopreservation. The results showed that without induced extracellular ice nucleation, the viability of spheroids containing 4,000 cells was only 26% after thawing, compared with 55% when PWW was used to induce nucleation. For spheroids containing 8,000, the PWW treatment improved viability from 45 to 76%. The researchers further examined the thawed spheroids using a live/dead cell assay, in which live cells were stained green and dead cells were stained red, and observed them using confocal microscopy. In the spheroids that were preserved using DMSO only, there were more red-stained cells throughout the assembly than in the PWW-treated spheroids, supporting the idea that inducing extracellular ice nucleation helped prevent cell death due to intracellular ice formation. This study was published in JACS Au.
“Cryopreservation is essential across all biomedical research and drug discovery. Most people assume cryopreservation is focussed on stopping ice forming, but it is actually essential to help ice form, at the highest temperature possible,” said co-corresponding author Thomas Whale. “We achieved this, and show that by helping the ice form we can dramatically improve the recovery of these cells.”
As PWW is soluble and easily sterilized with filtration methods, it presents a promising tool for protecting complex cell models from fatal ice formation. The authors note that ice nucleating molecules that promote ice formation at even higher temperatures could further improve cell recovery from cryopreservation in the future.