Non-uniform distribution of cryoprotecting agents in rice culture cells measured by CARS microscopy

Authors: SAMUELS, FIONNA - Colorado State University; STICH, DOMINIK - University Of Colorado; Bonnart, Remi; Volk, Gayle; LEVINGER, NANCY - Colorado State University

Submitted to: Plants
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/18/2021
Publication Date: 3/21/2021
Citation: Samuels, F.M., Stich, D.G., Bonnart, R.M., Volk, G.M., Levinger, N.E. 2021. Non-uniform distribution of cryoprotecting agents in rice culture cells measured by CARS microscopy. Plants. 10(3):589. https://doi.org/10.3390/plants10030589.
DOI: https://doi.org/10.3390/plants10030589

Interpretive Summary: Cryopreservation has been used to successfully preserve plant cells and shoot tips for long-term conservation within genebanks. Successful shoot tip cryopreservation usually depends on the use of cryoprotectant solutions that dehydrate cells and replace cellular water with other chemical compounds, such as dimethyl sulfoxide (DMSO). DMSO is believed to enter plant cells during cryoprotectant treatment. This research, performed in collaboration with Colorado State University, used Coherent anti-Stokes Raman scattering (CARS) microscopy to visualize deuterated DMSO within rice suspension cells. This work showed that d6-DMSO does not uniformly distribute throughout the cells, and instead enters the cell and sequesters within plastids. This changes our understanding of how DMSO concentration varies within the cellular compartments. Variations in cryoprotectant concentration across different cells and tissues will likely lead to differing protection from liquid nitrogen exposure. Expanding this work to include different cryoprotectants and mixtures of cryoprotectants is vital to create a robust understanding of how the distribution of these molecules change when different cryoprotectants are used.

Technical Abstract: Cryoprotectants allow cells to be frozen and cryopreserved for years by minimizing damages that occur in freezing and thawing processes. Unfortunately, how the specific cryoprotectants keep the cells viable through the cryopreservation process is not evident. This lack of fundamental understanding contributes to the arduous process of optimizing cryoprotectant formulations for each new cell line or species that is preserved, as there are not clear guidelines for how each cryoprotectant contributes to protecting the cell. Coherent anti-Stokes Raman scattering microscopy facilitates the visualization of deuterated cryoprotectants within living cells. Using this technique, we directly imaged the location of fully deuterated dimethyl sulfoxide (d6-DMSO), the deuterated form of a commonly used cryoprotectant, DMSO, within rice suspension cells. This work showed that d6-DMSO does not uniformly distribute throughout the cells, rather entering the cell and sequestering within organelles, changing our understanding of how DMSO concentration varies within the cellular compartments. Variations in cryoprotectant concentration across different cells and tissues will likely lead to differing protection from liquid nitrogen exposure. Expanding this work to include different cryoprotectants and mixtures of cryoprotectants is vital to create a robust understanding of how the distribution of these molecules change when different cryoprotectants are used.