Carbohydrate-mediated inhibition of ice recrystallization in cryopreserved human umbilical cord blood

Cryopreservation of human umbilical cord blood (UCB) typically involves the cryoprotectant dimethylsulfoxide (DMSO), however, infusional toxicity and reductions in cell viability remain a concern. Ice recrystallization (IR) is an important source of cryopreservation-induced cellular injury and limits the stem cell dose in UCB units. Carbohydrates have wide-ranging intrinsic IR inhibition (IRI) activity related to structural properties. We investigated the impact of carbohydrate IRI on cell viability, induction of apoptosis and hematopoietic progenitor function in cryopreserved UCB. Mononuclear cells (MNCs) from UCB were cryopreserved in storage media containing specific carbohydrates (200 mM) and compared to 5% DMSO. Samples were analyzed under conditions of high IR (‘slow’ thaw) and low IR (‘fast’ thaw). Thawed samples were analyzed for viability and apoptosis by flow cytometry and hematopoietic function using colony-forming unit (CFU) assays. IRI of carbohydrate solutions was determined using the ‘splat cooling’ assay. Greater IRI capacity of carbohydrates correlated with increased yield of viable MNCs (r2 = 0.92, p = 0.004) and CD34(+) cells (r2 = 0.96, p = 0.019) after thawing under conditions of high IR. The correlations were less apparent under conditions of low IR. Carbohydrates with greater IRI modulate the induction of early apoptosis during thawing, especially in CD34+ cells (r2 = 0.96, p = 0.0001) as compared to total mononuclear cells (p = 0.006), and preserve CFU capacity in vitro (r2 = 0.92, p = <0.0001). Our results suggest that carbohydrates with potent IRI increase the yield of non-apoptotic and functional hematopoietic progenitors and provide a foundation for the development of novel synthetic carbohydrates with enhanced IRI properties to improve cryopreservation of UCB.

Graphical abstractIce recrystallization is inhibited by natural carbohydrates, as revealed by light microscopy of thawed phosphate buffered saline solution (left) compared with 200 mM melibiose solution (right). Human umbilical cord blood mononuclear cells cryopreserved in melibiose have reduced levels of apoptosis and greater yield of functional hematopoietic precursors after thawing compared with cells cryopreserved using carbohydrates with less potent inhibition of ice recrystallization.Figure optionsDownload full-size imageDownload as PowerPoint slide