Human leukemia cells (HL-60) proteomic and biological signatures underpinning cryo-damage are differentially modulated by novel cryo-additives.

BACKGROUND: Cryopreservation is a routinely used methodology for prolonged storage of viable cells. The use of cryo-protective agents (CPAs) such as dimethylsulfoxide (DMSO), glycerol, or trehalose is paramount to reducing cellular cryo-injury, but their effectiveness is still limited. The current study focuses on establishing and modulating the proteomic and the corresponding biological profiles associated with the cryo-injury of human leukemia (HL-60) cells cryopreserved in DMSO alone or DMSO +/- novel CPAs (e.g., nigerose [Nig] or salidroside [Sal]). FINDINGS: To reduce cryo-damage, HL-60 cells were cultured prior and post cryopreservation in malondialdehyde Roswell Park Memorial Institute medium-1640 media +/- Nig or Sal. Shotgun proteomic analysis showed significant alterations in the levels of proteins in cells cryopreserved in Nig or Sal compared to DMSO. Nig mostly affected cellular metabolism and energy pathways, whereas Sal increased the levels of proteins associated with DNA repair/duplication, RNA transcription, and cell proliferation. Validation testing showed that the proteome profile associated with Sal was correlated with a 2.8-fold increase in cell proliferative rate. At the functional level, both Nig and Sal increased glutathione reductase (0.0012±6.19E-05 and 0.0016±3.04E-05 mU/mL, respectively) compared to DMSO controls (0.0003±3.7E-05 mU/mL) and reduced cytotoxicity by decreasing lactate dehydrogenase activities (from -2.5 to -4.75 fold) and lipid oxidation (-1.6 fold). In contrast, only Nig attenuated protein carbonylation or oxidation. CONCLUSIONS: We have identified key molecules and corresponding functional pathways underpinning the effect of cryopreservation (+/- CPAs) of HL-60 cells. We also validated the proteomic findings by identifying the corresponding biological profiles associated with promoting an anti-oxidative environment post cryopreservation. Nig or Sal in comparison to DMSO showed differential or additive effects in regard to reducing cryo-injury and enhancing cell survival/proliferation post thaw. These results can provide useful insight to cryo-damage and the design of enhanced cryomedia formulation.

Cryopreservation of Red Blood Cells.

Glycerol and trehalose are widely used protective agents in the cryopreservation of red blood cells (RBCs). This chapter presents a protocol for use of these agents as cryoprotectants of RBCs, with post-thaw assessment of cell survival and cellular oxidative-reductive status. The main aim is to provide a framework for further studies aimed at improving RBC survival and function and to supply improved biomaterials for studies on RBC biochemistry, major operations, as well as those for urgent use in emergency room situations.

Molecular Characterization of Human Leukemia 60 (HL-60) Cells as a Model of Acute Myelogenous Leukemia Post Cryopreservation.

The use of human leukemic (HL)-60 cells is important for studies of acute myeloid leukemia (AML) and as a model system for investigating how specific types of blood cells are formed during the process of hematopoiesis. Here, we present a protocol for growth of HL-60 cells along with molecular and functional profiles associated with their cryostorage. We also elucidate the effects of these procedures on cell viability and functions. This method can be used to provide biomarkers as readouts for testing the efficacy and/or toxicity of novel compounds in AML research as well as in a number of other experimental manipulations.