Novel Cryoprotective agents to improve the quality of cryopreserved mammalian cells

Abstract

Cryopreservation is a promising approach to long-term biopreservation of living cells, tissues and organs. The use of cryoprotective agents (CPAs) in combination with extremely low temperatures is mandatory for optimum biopreservation. CPAs (e.g., glycerol, trehalose, dimethyl sulphoxide (DMSO)), however, are relatively cytotoxic and compromise biopreserved cell quality. This is usually resultant in oxidative damage, diminishing cell functionality and survival rate. The growing market of cell therapy medicinal products (CTMPs) demands effective cryopreservation with greater safety, of which the currently available CPAs are unable to provide. The present study was aimed at developing cryomedia formulation to enhance the cryopreservation of nucleated and anucleated mammalian cells. Here, eleven compounds of a polyol nature were selected and examined for their cryoprotective properties. These compounds are derived from plants and honey, thereby ensuring their safety for human consumption. The selection was based on their molecular structure and chemical properties. Here, the presented study is divided into three main phases: 1) Screening the compounds panel for cryo-additive effects on cells during and post-cryopreservation and optimising the dose response and time course for trehalose and glycerol with and without the novel compounds; 2) Assessing the influence of biophysical criteria on biospecimen cryopreservation (e.g., biosampling procedure, cell age, donor age); 3) Establishing the mechanisms of action underpinning the modulatory effect of novel CPAs on biological pathways during cryopreservation. For the stated purposes, red blood cells (RBCs) obtained from sheep and humans were used to screen the compounds for novel cryo-additive agents. Cryosurvival rate was employed as an indication of the compounds’ cryoprotective performance. Cellular biochemical profiles, including lipid and protein oxidative damage as well as key redox enzymatic activities (e.g., lactate dehydrogenase (LDH), glutathione reductase (GR)) were measured. The study revealed that nigerose (Nig) and salidroside (Sal) were significantly effective in protecting cells during the freeze-thaw cycle and recovery phases. Both compounds promoted the activity of GR and reduced oxidative stress mirrored by diminished LDH activity. This was also reflected in the protein and lipid oxidation levels, which was limited to a comparable level with the cells’ prior freezing. Further studies on human leukaemia (HL-60) were carried out to elucidate the molecular and biological pathways associated with cryodamage and the modulatory effects of adding novel CPAs. The proteome profile and the corresponding biological functions were evaluated and iii showed that Nig and Sal protected cells against cryodamage. The additive compounds (Nig and Sal) demonstrated a unique and overlapping modulation effect pattern. Nig was found to highly influence proteins engaged with metabolic and energetic pathways, whereas Sal greatly affected nuclear and DNA-binding proteins. The current study concluded that novel CPAs have high potency in protecting cells and each compound has a unique effect on the cellular proteome. These features can be applied to designing cryomedia formulae with higher protective efficiency for targeted applications in cell based therapy and biopharmaceutical industries.