Clinical approaches in regenerative medicine frequently involve the transplantation of cells, tissue constructs, and organs [31]. While many studies have worked to optimize the culturing of live cells prior to transplantation [32–34], a major obstacle in this field is the distribution and timing of finite products for clinical use [35]. As allogenic products are becoming more frequently used, it is paramount to optimize the long-term storage of cells and tissues to help minimize clinical costs and make the distribution easier.
We and others have demonstrated that the successful cryopreservation of different human cell types can be accomplished using HES alone or in combination with DMSO [16]. It was previously shown by Pasch et al. that HES can be used to optimally cryopreserve human keratinocytes in suspension and in monolayers [20, 21]. Our data for HaCaT cells corroborate these findings, showing that both cell numbers (Fig. 1) and cellular structures (Fig. 8) are comparable to fresh cells following cryopreservation with HES. Table 3 summarizes the optimal cryopreservation solution containing HES identified for each cell type. For keratinocytes cryopreserved in either suspension or in monolayers, both the 5% DMSO, 5% HES, 90% FCS and the 10% HES, 90% FCS solutions maintained cell viability (Table 3).
The optimal cryopreservation solutions containing HES identified for keratinocytes and fibroblasts
We previously identified the ideal freezing protocols to use for the cryopreservation of human keratinocytes and fibroblasts [30]. To our knowledge, however, no group has compared cryopreservation efficacy in fibroblasts preserved with HES versus other CPAs. As assessed by measuring cell numbers (Fig. 2) and analyzing nuclei, actin, and mitochondria (Fig. 8), cryopreservation of BJ fibroblasts in suspension was successfully achieved using a 5% DMSO, 5% HES, 90% FCS or a 10% HES, 90% FCS solution (Table 3). BJ cells in monolayers were cryopreserved well in a 5% DMSO, 5% HES, 90% FCS solution (Table 3). In contrast, primary fibroblasts in monolayers showed decreased cell viability when cryopreserved in any solution containing HES (Figs. 3 and 8). Primary fibroblasts in suspension could be successfully cryopreserved in a 5% DMSO, 5% HES, 90% FCS solution (Table 3).
Significant differences in cell viability were observed between HaCaT, BJ, and primary fibroblast cells (Figs. 1, 2, and 3). During cryopreservation, cells and tissues must traverse a lethality zone of temperature (?15 to ?60 °C) when being frozen down to very low temperatures and then once again when being thawed [36]. While passing through this zone, cells and tissues must endure the damaging effects of vitrification, cold shock, osmotic injury, and intracellular ice formation [36]. Our data indicate that different cell types are uniquely capable of resisting and repairing damage that occurs during cryopreservation and subsequent thawing. Of particular interest is that the viability of BJ cell line fibroblasts (Fig. 2) was notably higher than the viability of primary fibroblasts (Fig. 3). One possibility is that, since BJ cells are commercially available and routinely undergo freeze-thaw cycles, this cell line has undergone a selection for cells that can robustly withstand cryopreservation. In contrast, primary fibroblasts derived from donors have had no such opportunity to undergo a selection for cells especially resistant to freezing-induced damage. HaCaT cells seemed to cryopreserve more efficiently than fibroblast cells. Since keratinocytes are smaller in size than fibroblasts [37, 38] and cells tend to shrink due to osmosis during cryopreservation [39], a potential explanation for this is difference is that fibroblasts undergo more drastic cell shrinkage during freezing and subsequent thawing. This proposed increase in cell shrinkage stress may result in decreased viability.
As we have previously found with mesenchymal stem cells [29], predominantly negligible differences in cell viability were found immediately post-thawing at Day 0 for HaCaT, BJ, and primary fibroblast cells (Fig. 4). This is most likely explained by the fact that cryopreservation-induced apoptotic cell death has been estimated to take between 6 and 24 h [40] and can still be observed 24-h post-thaw [41]. We chose not to include additional time points beyond Day 3 as, in our previous work with mesenchymal stem cells [29], we found minimal differences in cell viability between Day 3 and Day 14-post-thaw. Interestingly, the cell number for some cryopreservation conditions in HaCaT cells was higher than the control, fresh cells (Fig. 1). It is possible that, for certain cell types, specific CPA solutions promote viability and growth over fresh cells. The general variations in cell number are due to each cell type having a unique proliferation and survival rate in adherent vs. suspension conditions.
It should be noted that, while HES is thought to be clinically safe [16], toxic effects of HES have been noted in the literature. Singbartl et al. reported that cryopreservation of erythrocytes using HES in lieu of glycerol led to the development of transient rheological alterations, including an altered membrane skeleton [42]. A systematic review assessing the use of HES for fluid management found that HES may increase the risk of acute renal failure in patients with sepsis [43]. This risk appeared to increase with higher doses of HES [43]. While HES has been reported in association with adverse effects, a much higher of concentration of HES is required to induce toxicity than DMSO [16].
A randomized phase III clinical trial found that autologous blood stem cell transplantation could be effectively accomplished using cells cryopreserved in a 5% DMSO, 6% HES solution or a 10% DMSO solution. While two patients infused with cells cryopreserved with DMSO displayed serious neurological toxicity, none of the patients who received DMSO/HES cryopreserved cells showed any serious toxicities [44]. Given concerns of DMSO toxicity and the low cost of HES, the authors of that trial suggested replacing a portion of DMSO with HES when cryopreserving cells [44]. A literature review summarizing the known data regarding HES corroborates this trial data, suggesting that HES is less toxic than DMSO and that, at low concentrations, HES is clinically safe [16]. Although the data seem to suggest that DMSO is more toxic than HES, further studies should be performed to specifically compare the clinical safety of HES vs DMSO in patients transplanted or infused with cryopreserved cells.
While we feel that the data contained herein are novel and significant, there are limitations of this work. Ideally, the concentration of FCS would be decreased or FCS would be omitted entirely since it is a derived animal product and therefore susceptible to contamination. Although we did include two different CPA combinations that omitted FCS entirely (10% HES, 90% DMEM and 10% DMSO, 90% DMEM), the remaining CPA combinations only featured two different concentrations of FCS – 90% and 95%. Future studies are warranted to assess whether or not such high concentrations of FCS are required when using HES as a CPA. Moreover, it would be of interest to measure cell viability and cryopreservation efficiency by investigating other parameters, such as cell viability, recovery, and metabolism. Growth rates, cell-substrate attachment, and gene and protein analyses would also be valuable to quantify. While we did not assess these parameters in this manuscript, we are interested in building off the present study to do so with our future investigations.