Polyethylene glycol 400 enables plunge-freezing cryopreservation of human keratinocytes

Abstract

An effort was made to design a simple and efficient protocol for the cryopreservation of human keratinocytes, a cell line relevant to tissue engineering. A possibility of simultaneously preventing the injury from intracellular ice formation and slow-freezing injury by introducing a macromolecular non-permeant polyethylene glycol 400 as a sole cryoprotectant with rapid freezing was recognized. Thermoanalytical and microstructural analysis of the potential cryoprotective mixture has been performed to test its efficacy in preventing cell cryoinjury mechanisms. The post-thaw cell recovery indicated successful cryopreservation with a similar recovery to the standard slow-freezing protocol with permeant dimethyl sulfoxide. The novel approach represents an alternative cryopreservation strategy that avoids intracellular cryoprotectant toxicity and offers a simple freezing procedure (plunging into liquid nitrogen) and a more practical cryoprotectant wash-out after the thawing. The cryopreservation protocol also brings up new possibilities for applying macromolecular additives as novel cryoprotectants.