Cryopreservation Effects on Wharton’s Jelly Stem Cells Proteome

Abstract

Cryopreservation is the only method for long-term storage of viable cells and tissues used for cellular therapy, stem cell transplantation and/or tissue engineering. However, the freeze-thaw process strongly contributes to cell and tissue damage through several mechanisms, including oxidative stress, cell injury from intracellular ice formation and altered physical cellular properties. Our previous proteomics investigation was carried out on Wharton's Jelly Stem Cells (WJSCs) having similar properties to adult mesenchymal stem cells and thus representing a rich source of primitive cells to be potentially used in regenerative medicine. The aim of the present work was to investigate molecular changes that occur in WJSCs proteome in different experimental conditions: fresh primary cell culture and frozen cell. To analyze changes in protein expression of WJSCs undergoing different culturing procedures, we performed a comparative proteomic analysis (2DE followed by MALDI-TOF MS/MS nanoESI-Q-TOF MS coupled with nanoLC) between WJSCs from fresh and frozen cell culturing, respectively. Frozen WJSCs showed qualitative and quantitative changes compared to cells from fresh preparation, expressing proteins involved in replication, cellular defence mechanism and metabolism, that could ensure freeze-thaw survival. The results of this study could play a key role in elucidating possible mechanisms related to maintaining active proliferation and maximal cellular plasticity and thus making the use of WJSCs in cell therapy safe following bio-banking.

Umbilical Cord Tissue-Derived Cells as Therapeutic Agents

“…Other papers gave alternative methods. Di Giuseppe et al [ 114 ] carried out profound research on the cryopreservation effects on WJSCs proteome. It was demonstrated that frozen WJSCs showed qualitative and quantitative changes compared to fresh cells, expressing proteins involved in replication, cellular defence mechanism, and metabolism, which could ensure freeze-thaw survival.…”

Diameters and Fluorescence Calibration for Extracellular Vesicle Analyses by Flow Cytometry

“…We established the value for the trigger threshold on the PE (Phycoerythrin) channel which did not produce CD90+-EV loss, in order to measure the resolution sensitivity of the flow cytometer on the basis of PE-conjugated to the anti-CD90 which is a well-recognized MSC marker also expressed on MSC-derived EVs [ 39 , 40 , 41 , 42 ]. Three different samples of MSC-derived EVs (obtained from three different MSC clones) were then acquired triggering on side scatter (SSC) or on PE (CD90 detection channel).…”

Directional freezing for the cryopreservation of adherent mammalian cells on a substrate

“…Although cryopreservation techniques are widely used, emerging data suggest that cryopreservation procedures affect cell survival, cell function, protein expression, and DNA integrity, as well as the cytoskeletal and nuclear structures [ 7 , 38 – 41 ]. Common CPAs, such as DMSO, have been shown to display toxic effects on cells that are considered to be temperature-, time-, and concentration-dependent [ 39 , 42 , 43 ].…”

“…It is thus become apparent that reducing the exposure to CPAs may be critical for improving in-vitro models of disease used in mechanistic and drug development research, as well as in the emerging field of stem cell research. Several studies have shown that cryopreserving stem cells can reduce pluripotency marker expression and reduce the cells’ capacity to differentiate into specific lineages or, alternatively, to trigger spontaneous differentiation [ 38 , 44 – 48 ]. These features may be cell type specific [ 49 ]; therefore, the development of successful cryopreservation methods that reduce the local CPA exposure of the cells, for example by applying directional freezing, is a required step for advancing a wide range of cell-based in vitro models.…”