Characterizing cellular membrane partitioning of DMSO using low-temperature Raman spectroscopy

Area of Science:

• Cellular biology
• Biophysics
• Cryobiology

Background:

• Cryoprotective agents (CPAs) are essential for cell survival during freezing and thawing.
• Understanding CPA location (intracellular vs. extracellular) is key to their mechanism of action.
• Low-temperature Raman spectroscopy offers label-free, high-resolution detection of CPA distribution.

Purpose of the Study:

• To determine the partitioning ratio of dimethylsulfoxide (DMSO) in cryopreserved cells using Raman spectroscopy.
• To investigate how cooling rates and DMSO concentrations affect DMSO partitioning.
• To explore cell-type specific differences in DMSO partitioning during cryopreservation.

Main Methods:

• Cells were cryopreserved using varying cooling rates and DMSO concentrations.
• Low-temperature Raman spectroscopy was employed to image CPA location.
• Automated image analysis quantified the DMSO partitioning ratio (extracellular/intracellular concentration).

Main Results:

• Jurkat cells showed a partitioning ratio near 1 at 1°C/min, except for 1% DMSO (ratio 0.2).
• Increasing cooling rate from 1°C/min to 5°C/min increased the partitioning ratio from 1 to 1.3.
• Sensory neurons and iPSCs exhibited different partitioning ratios, indicating cell-type specific responses.
• Intracellular ice formation altered DMSO distribution and the partitioning ratio.

Conclusions:

• DMSO partitioning is influenced by cooling rate, concentration, and cell type.
• Raman spectroscopy effectively quantifies CPA distribution, aiding cryopreservation optimization.
• Cellular differences in solute partitioning may explain variations in freezing tolerance.