Cryopreservation of Stem Cells Using Trehalose: Evaluation of the Method Using a Human Hematopoietic Cell Line

Abstract

While stem cell cryopreservation methods have been optimized using dimethylsulfoxide (DMSO), the established techniques are not optimal when applied to unfertilized human embryonic cells. In addition, important questions remain regarding the toxicity and characteristics of DMSO for treatment of stem cells for clinical use. The objective of this study was to establish an optimal method for cryopreservation of stem cells using low concentrations (0.2 M) of trehalose, a nontoxic disaccharide of glucose, which possesses excellent protective characteristics, in place of current methods utilizing high concentrations (1-2 M) of DMSO. A human hematopoietic cell line was used in this investigation as a surrogate for human stem cells. Trehalose was loaded into cells using a genetically engineered mutant of the pore-forming protein alpha-hemolysin from Staphylococcus aureus. This method results in a nonselective pore equipped with a metal-actuated switch that is sensitive to extracellular zinc concentrations, thus permitting controlled loading of trehalose. Preliminary experiments characterized the effects of poration on TF-1 cells and established optimal conditions for trehalose loading and cell survival. TF-1 cells were frozen at 1 degrees C/min to -80 degrees C with and without intra- and extracellular trehalose. Following storage at -80 degrees C for 1 week, cells were thawed and evaluated for viability, differentiation capacity, and clonogenic activity in comparison to cells frozen with DMSO. Predictably, cells frozen without any protective agent did not survive freezing. Colony-forming units (CFU) generated from cells frozen with intra- and extracellular trehalose, however, were comparable in size, morphology, and number to those generated by cells frozen in DMSO. There was no observable alteration in phenotypic markers of differentiation in either trehalose- or DMSO-treated cells. These data demonstrate that low concentrations of trehalose can protect hematopoietic progenitors from freezing injury and support the concept that trehalose may be useful for freezing embryonic stem cells and other primitive stem cells for therapeutic and investigational use.

Evaluation of Methylcellulose and Dimethyl Sulfoxide as the Cryoprotectants in a Serum-Free Freezing Media for Cryopreservation of Adipose-Derived Adult Stem Cells

“…Although DMSO is regarded as relatively nontoxic, the clinical use of frozen/thawed cells treated with DMSO can cause many adverse effects and toxic reactions [7][8][9][10][11][12][13][14]. It has also been reported that DMSO is not only cytotoxic but it also induces differentiation of stem cells to cardiac or neuronal like cells when added to the cell culture medium [15,16].…”

“…It is, therefore, imperative to reduce the toxicity by the removal of DMSO prior to clinical use. However, the total removal of DMSO from the frozen/thawed cells is costly and time consuming and invariably results in cell loss and clumping [7,8]. Therefore, it is necessary to develop cryopreservation protocols either with lower concentrations of DMSO or with nontoxic alternatives to DMSO.…”

Cryoprotective Effect of Disaccharides on Cord Blood Stem Cells with Minimal Use of DMSO

“…These cells also retained the potential to multilineage engraftment in nonobese diabetic-SCID mice [22]. Extracellular and intracellular loaded trehalose also proved to be an effective CPA for HSCs as suggested by one of the lab report [23]. Trehalose, and to some extent sucrose can stabilize proteins as well as lipid bilayer.…”

Is stem cell chromosomes stability affected by cryopreservation conditions?

“…Trehalose forms a glass, a crucial process in cell preservation and storage in a dry state even at high temperatures and moisture content. The major problem in using this disaccharide is the impermeability of the plasma membrane leading to considerable difficulty in introducing high concentrations of the polymer in the cell cytoplasm (Buchanan et al 2004). Major manipulations, like cell permeabilization, need to take place first, but this may affect signalling pathways due to cell receptor and membrane channel damages.…”