Investigating solution effects injury of human T lymphocytes and its prevention during interrupted slow cooling

Cooling rate is a critical parameter affecting the success of cell cryopreservation. Fast cooling can result in intracellular ice formation (IIF), while slow cooling can bring solution effects injury, both are detrimental to the cells. Whilst most of the studies have investigated how IIF affects cells, solution effects injury has received little attention. Here, we studied the solution effects injury of human T lymphocytes by cryomicroscopy and tested the osmoprotective ability of some frequently used cryoprotective agents (CPAs) such as dimethyl sulfoxide (DMSO), glycerol, trehalose, urea and l-proline. We further investigated the relationship between cell volume, latent heat and solution effects cell injury. We found that solution effects injury during interrupted slow cooling was caused by high concentration of the extracellular solution rather than eutectic formation and solutes precipitation. DMSO, glycerol and trehalose can protect cells from solution effects injury, while l-proline and urea cannot under the same condition. The cell volume and latent heat are not crucial for causing solution effects injury in cells. This work confirms that high osmotic pressure, rather than eutectic formation, leads to cell injury. It also suggests that cell volume and latent heat may not be a key factor for explaining solution effects injury and its prevention in the cryopreservation of human T lymphocytes.