Cryopreservation of dermal fibroblasts and keratinocytes in hydroxyethyl starch–based cryoprotectants

Abstract

BackgroundPreservation of human skin fibroblasts and keratinocytes is essential for the creation of skin tissue banks. For successful cryopreservation of cells, selection of an appropriate cryoprotectant agent (CPA) is imperative. The aim of this study was to identify CPAs that minimize toxic effects and allow for the preservation of human fibroblasts and keratinocytes in suspension and in monolayers.ResultsWe cryopreserved human fibroblasts and keratinocytes with different CPAs and compared them to fresh, unfrozen cells. Cells were frozen in the presence and absence of hydroxyethyl starch (HES) or dimethyl sulfoxide (DMSO), the latter of which is a commonly used CPA known to exert toxic effects on cells. Cell numbers were counted immediately post-thaw as well as three days after thawing. Cellular structures were analyzed and counted by labeling nuclei, mitochondria, and actin filaments. We found that successful cryopreservation of suspended or adherent keratinocytes can be accomplished with a 10% HES or a 5% HES, 5% DMSO solution. Cell viability of fibroblasts cryopreserved in suspension was maintained with 10% HES or 5% HES, 5% DMSO solutions. Adherent, cryopreserved fibroblasts were successfully maintained with a 5% HES, 5% DMSO solution.ConclusionWe conclude that skin tissue cells can be effectively cryopreserved by substituting all or a portion of DMSO with HES. Given that DMSO is the most commonly used CPA and is believed to be more toxic than HES, these findings are of clinical significance for tissue-based replacement therapies. Therapies that require the use of keratinocyte and fibroblast cells, such as those aimed at treating skin wounds or skin burns, may be optimized by substituting a portion or all of DMSO with HES during cryopreservation protocols.

Freshly Thawed and Continuously Cultured Human Bone Marrow-Derived Mesenchymal Stromal Cells Comparably Ameliorate Allergic Airways Inflammation in Immunocompetent Mice

“…In the current study, cell viability was ∼70% after thawing and harvest compared with ∼100% viability of the freshly harvested, continuously cultured cells. These are comparable to experiences in other studies, as are the freezing medium and the freeze-thaw technique used [11][12][13][14]. As such, there is no obvious discrepancy in the technical approaches.…”

“…The frozen MSCs, suspended at the desired concentration in a cryopreservation medium suitable for infusion, are then thawed just prior to administration. However, a significant number of the frozen MSCs may undergo apoptosis during the freeze-thaw process, although much subsequent study has attempted to minimize this occurrence with improved freeze-thaw approaches and cryopreservatives [11][12][13][14]. A recent report demonstrated that freshly thawed MSCs were not as effective in in vitro potency assays as continuously cultured MSCs of the same passage number [15].…”

“…The length of freezing prior to thaw in the current studies was relatively short (7 days) and may not have had the same effect as longer freezing. Longer freeze time prior to thawing and use has been demonstrated to have detrimental effects on initial cell viability after thawing and on proliferative and differentiation capacities [11][12][13][14]. However, length of freeze time on anti-inflammatory actions is not yet well understood.…”

Human cultured epidermis accelerates wound healing regardless of its viability in a diabetic mouse model

“…Although the consistency of allo-CE was preserved by being stored in culture medium at 22-24˚C for 20 days [17] or cryopreserved at -80˚C in culture medium containing fetal calf serum and glycerol as a cryoprotectant [6], the cell viability of the keratinocytes was depleted in any case. When cells and tissues are cryopreserved, they are damaged by vitrification, cold shock, osmotic injury, and intracellular ice formation [21,22].…”

The Effect of Endothelial Cells on UVB‐induced DNA Damage and Transformation of Keratinocytes In 3D Polycaprolactone Scaffold Co‐culture System

“…B) while the 2D cultured cells were irregularly shaped (Data not shown). Using freshly thawed HaCaT cells to mimic cells freshly isolated from tissues , we determined whether the 3D cultured cells are more similar to cells freshly isolated from tissues than the 2D cultured cells by comparing vimentin expression in 2D and 3D cultured HaCaT cells. Vimentin is a biomarker for mesenchymal and indicates the differences in the composition of cell cytoskeleton and the extracellular matrix (ECM) between 2D and 3D cells .…”