Inhibiting ice recrystallization and optimization of cell viability after cryopreservation

Abstract

The ice recrystallization inhibition activity of various mono- and disaccharides has been correlated with their ability to cryopreserve human cell lines at various concentrations. Cell viabilities after cryopreservation were compared with control experiments where cells were cryopreserved with dimethylsulfoxide (DMSO). The most potent inhibitors of ice recrystallization were 220 mM solutions of disaccharides; however, the best cell viability was obtained when a 200 mM d-galactose solution was utilized. This solution was minimally cytotoxic at physiological temperature and effectively preserved cells during freeze-thaw. In fact, this carbohydrate was just as effective as a 5% DMSO solution. Further studies indicated that the cryoprotective benefit of d-galactose was a result of its internalization and its ability to mitigate osmotic stress, prevent intracellular ice formation and/or inhibit ice recrystallization. This study supports the hypothesis that the ability of a cryoprotectant to inhibit ice recrystallization is an important property to enhance cell viability post-freeze-thaw. This cryoprotective benefit is observed in three different human cell lines. Furthermore, we demonstrated that the ability of a potential cryoprotectant to inhibit ice recrystallation may be used as a predictor of its ability to preserve cells at subzero temperatures.

Small molecule ice recrystallization inhibitors enable freezing of human red blood cells with reduced glycerol concentrations

“…In fact, b-PMP-Glc (6) exhibited activity similar to the previously reported non-ionic surfactant b-Octyl-Gal (1) at equimolar concentration 25 . b-PMP-Glc (6) also exhibited comparable IRI activity to a 220 mM solution of sucrose, which is a common ingredient of many cryopreservation solutions 28 . Our laboratory has previously correlated IRI activity to carbohydrate hydration and reported that glycoconjugates containing highly hydrated monosaccharides (such as D-galactose) were more IRI active than those containing less hydrated monosaccharides (D-glucose and D-mannose) 23,43 .…”

“…NOGlc could not be assessed in the freezing assays at concentrations greater than 5 mM due poor solubility in aqueous solutions containing glycerol 25,49 . Sucrose was examined at concentrations of 220 mM and 500 mM as it has previously been identified as an effective inhibitor of ice recrystallization and can effectively cryopreserve human embryonic liver cells 28 . Solutions containing RBCs were cooled slowly at a rate of 1uC/min to 25, 215 or 240uC and then immersed in liquid nitrogen (2196uC) before being thawed.…”

“…Our laboratory has recently reported the rational design and synthesis of large Clinked glycoconjugates [22][23][24] as well as small molecules [25][26][27] that inhibit the process of ice recrystallization. We hypothesize that inhibiting this process will greatly reduce cellular injury during cryopreservation and increase the post-thaw recovery of cells with specialized cellular functions 24,28 . Current research on improving RBC cryopreservation has focused on a number of different aspects of the cryopreservation protocol in an attempt to overcome the limitations associated with using glycerol as a cryoprotectant.…”

Modulation of antifreeze activity and the effect upon post-thaw HepG2 cell viability after cryopreservation

“…HepG2 (human liver hepatocellular carcinoma cells, ATCC, HB-8065) were cultured in Eagle's minimum essential media (MEM) supplemented with 10% FBS (fetal bovine serum), 1% non-essential amino acids, 1 mM sodium pyruvate and 1% penicillin-streptomycin in 75 cm 2 Corning Ò flasks [14]. Cells were incubated in a 37°C incubator supplied with 5% CO 2 .…”

“…HepG2 cells were cultured and detached as described above [14]. Cells were diluted with culture media and counted using a hemocytometer.…”