Pre-incubation with hyaluronan reduces cellular damage after cryopreservation in densely cultured cell monolayers

Reduction of cellular damage in densely cultured cell monolayers after cryopreservation by pre-incubation with hyaluronan (HA) was investigated. Monolayers of human dermal fibroblasts were cultured for 24 h at a density of 0.5×104 or 5×104 cells/cm2. The following two experimental conditions were compared: cells incubated with or without 0.5% w/w HA solution for 6 h. Samples were frozen from 4 to -80°C at 0.3 or 3°C/min in a cryoprotectant solution containing 10% w/w dimethyl sulfoxide, cooled down below -185°C, and then thawed. Post-thaw cell viability was evaluated by the fluorescent double-staining technique using a fluorescence microscope, and cellular uptake of the fluorescein-isothiocyanate-labeled HA after pre-incubation was also observed. Cell viability decreased with increasing cell density at both cooling rates without preliminary HA incubation. However, cell viability did not decrease at either cooling rate with preliminary HA incubation. Cellular HA uptake was observed. Pre-incubation with HA reduces cellular damage in densely cultured cell monolayers.