Freezing of dendritic cells with trehalose as an additive in the conventional freezing medium results in improved recovery after cryopreservation

Abstract

BACKGROUND: Dendritic cell (DC) vaccination involves administration of multiple doses. Cryopreservation of tumor antigen-pulsed DCs can provide a ready to use vaccine source and eliminate the need of frequent withdrawal of the patient's blood for vaccine preparation. The aim of this study was to assess the effect of addition of trehalose in the freezing medium on the recovery of DCs after cryopreservation.STUDY DESIGN AND METHODS: DCs were generated from mononuclear cells from apheresis samples of healthy donors. For long-term storage of 6 months, cells were frozen with a rate-controlled programmable freezer and stored in liquid nitrogen. For short-term storage of 1 month, cells were frozen and stored at −80 C. DCs frozen with Iscove's Modified Dulbecco's Medium + 10% dimethyl sulfoxide + 20% fetal bovine serum served as the control group, while the test group was additionally supplemented with 50 μg/mL of trehalose. After revival of control and test DCs, they were assessed for viability, morphology, phenotype, and functions. RESULTS:The addition of trehalose to the conventional freezing medium helped to preserve the viability and functionality of DCs better than dimethyl sulfoxide alone in both long-and short-term cryopreservation. Trehalose also protected the mitochondrial membrane potential and cytoskeleton integrity of DCs, which are necessary for their functionality. Mediators of the intrinsic apoptotic pathway like Caspase-9 and Bim-1 were found to be low in the test. CONCLUSION: Supplementation of conventional freezing medium with trehalose results in better quality of DCs revived after cryopreservation. This finding could help improve DC vaccine preparation for cancer immunotherapy. ABBREVIATIONS: CFSE = carboxyfluorescein succinimidyl ester; CPAs = cryoprotective agents; CTL = cytotoxic T-lympocyte; DC = dendritic cell; DMSO = dimethyl sulfoxide; FBS = fetal bovine serum; HLA = human leukocyte antigen; IMDM = Iscove's Modified Dulbecco's Medium; NCCS = National Centre for Cell Science; PBS = phosphate-buffered saline; PCR = polymerase chain reaction; TMRE = tetramethylrhodamine, ethyl ester. From the

The roles of reactive oxygen species and antioxidants in cryopreservation

“…It remains unclear if the up-regulation of antioxidant genes after antioxidant treatment provides direct benefit, if any, to protect cells against cryopreservation-induced ROS damage. Trehalose [201,202] and BHT [203] were reported to reduce lipid peroxidation [204] in testicular tissue and spermatozoa, respectively, generally enhance total antioxidant capacity, improve cellular viability [204–207] and reduce apoptosis [205,207]. Decrease in mitochondrial membrane potential (Δψ) has been observed in cells stimulated by apoptotic stimulus [208–211], which is also seen in thawed cells after cryopreservation [65].…”

“…Such impairments or undesirable outcomes of cellular functionality have been partly improved through administration of antioxidants. These include ascorbic acid [221], astragalosides [232], taurine [222], hypotaurine [178], vitamin E [76], catalase [221], trehalose [205–207], combination of catalase with trehalose [233,234] as well as combination of BHT with ascorbic acid [235] to cell types such as mononuclear cells, pancreatic islets, germ cells, spermatozoa, dendritic cells, hepatocytes and hemopoietic cells (Table 1). Among these antioxidants, winter wheat lipocalins and peroxiredoxins obtained from wheat are especially notable.…”

Trehalose in Biomedical Cryopreservation–Properties, Mechanisms, Delivery Methods, Applications, Benefits, and Problems

“…Trehalose is commonly used as a CPA. , Using it as the sole CPA could also protect cells and tissues even when it is not present in the cell. , …”

Exosomes based advancements for application in medical aesthetics

“…The most effective disaccharide antifreeze for exosomes is trehalose, which is mentioned as the best alternative ( Nakanishi et al, 2020 ). Trehalose and other membrane stabilisers have been used to store labile proteins, vaccines, liposomes, and cryopreserve tissues and stem cells, thus it makes sense to utilise them to keep exosomes stable ( Zhang X. G. et al, 2015 ; Bosch et al, 2016 ; Shinde et al, 2019 ). Budgude et al added 25 mM trehalose to exosomes in PBS at −80°C and found that these cryopreserved exosomes could be taken up by hematopoietic stem cells as efficiently as freshly isolated exosomes and that trehalose was as effective forfreshly isolated exosomes ( Budgude et al, 2021 ).…”