Intracellular delivery of trehalose renders mesenchymal stromal cells viable and immunomodulatory competent after cryopreservation

Abstract

Trehalose is a nontoxic disaccharide and a promising cryoprotection agent for medically applicable cells. In this study, the efficiency of combining trehalose with reversible electroporation for cryopreservation of two types of human mesenchymal stromal cells was investigated: adipose-derived stromal cells, and umbilical-cord-derived stromal cells. Comparable results to standard dimethyl sulfoxide cryopreservation protocols were achieved, even without extensive electroporation parameters and protocol optimization. The presence of high extracellular trehalose resulted in comparable cell viabilities without and with electroporation. According to the determination of trehalose concentrations, 250 mM extracellular trehalose resulting in, 20 mM to 50 mM intracellular trehalose were sufficient for successful cryopreservation of cells. With electroporation, higher (i.e. 50 mM to 90 mM) intracellular trehalose was achieved after cryopreservation, although cell survival was not improved significantly. To evaluate the impact of electroporation and cryopreservation on cells, stress and immune-activation-related gene expression were analyzed. Electroporation and/or cryopreservation resulted in increased SOD2 and HSPA1A expression. Despite the increased stress response, the high up-regulation by mesenchymal stromal cells of immunomodulatory genes in the inflammatory environment was not affected. Highest expression was seen for the IDO1 and TSG6 genes. In conclusion, cryopreservation of mesenchymal stromal cells in trehalose results in comparable characteristics to their cryopreservation using dimethyl sulfoxide.

Long-term and short-term preservation strategies for tissue engineering and regenerative medicine products: state of the art and emerging trends

“…Another possibility is to force nonpermeating cryoprotectants to enter inside the cell before cryogenic preservation. Considering this, there were attempts at delivering intracellularly nonpermeating cryoprotectants through the use of electroporation ( 31 , 32 ) or nanoparticles ( 33 , 34 ). During electroporation, the permeability of the cell membrane is transiently increased, which allows trehalose to be loaded into cells.…”

“…During electroporation, the permeability of the cell membrane is transiently increased, which allows trehalose to be loaded into cells. Loading trehalose is effective but does not significantly improve cell viability when in comparison with incubation with trehalose without electroporation ( 31 ). Electroporation is considered very harsh to cells.…”

“…Electroporation is considered very harsh to cells. Besides activating some stress responses such as heat shock proteins ( 31 ), it also irreversibly damages the cellular membrane ( 32 ), compromising cell viability. The use of nanoparticles is less invasive than electroporation.…”

Synthesis of Trehalose by the Erythritol-Producing Yeast Yarrowia lipolytica Co-Displaying Maltooligosyltrehalose Synthase and Maltooligosyltrehalose Trehalohydrolase

“…Trehalose is a commonly occurring nonreducing disaccharide, composed of two glucose molecules linked by a stable α-1,1-glycosidic bond . It has an excellent protective effect on proteins, biofilms, and other macromolecules, which makes trehalose a highly effective and nontoxic cryoprotectant for enzymes, animal cells, and plant cells in the medical field . Before the 1990s, trehalose was mostly extracted from Saccharomyces cerevisiae, which has a high cost .…”

“…1 It has an excellent protective effect on proteins, biofilms, and other macromolecules, which makes trehalose a highly effective and nontoxic cryoprotectant for enzymes, animal cells, and plant cells in the medical field. 2 Before the 1990s, trehalose was mostly extracted from Saccharomyces cerevisiae, which has a high cost. 3 Although trehalose can be chemically synthesized, its low yield and byproducts prevent it from being used in industrial production.…”