Changes in the Intracellular Composition of Macro and Microminerals After Cryopreservation of the Rabbit Stem/Progenitor Cells

Abstract

Cryopreservation is a widely used method for the long-term preservation of reproductive or somatic cells. It is known that this storage method may negatively affect cell viability, proliferation, differentiation, etc. However, there is a lack of information about whether cryostorage can alter the content of intracellular minerals. Therefore, we focused this study on the analysis of the mineral composition of living cells before and after long-term cold storage. Briefly, three different primary cell lines were established from rabbits as follows: endothelial progenitor cells from peripheral blood (EPCs), endothelial progenitor cells from bone marrow (BEPCs), and mesenchymal stem cells from adipose tissue (AT-MSCs), which were cultured until passage 3 prior to cryopreservation in liquid nitrogen. Samples from freshly cultured and frozen-thawed cells were mineralized and analyzed using inductively coupled plasma-optical emission spectroscopy (ICP-OES) for the content of minerals (macro: Ca, Na, K, and Mg, and micro: Zn, Fe, Cu, Al, Co, Mn, Sr, and Ni). After cryopreservation, we found significantly decreased content of K in frozen-thawed EPCs (p < 0.01) and BEPCs (p < 0.0001) and Ca in AT-MSCs (p < 0.05), while Na was increased in frozen-thawed BEPCs (p < 0.05). Concentrations of Fe and Al were reduced significantly in frozen-thawed EPCs (both p < 0.0001) and AT-MSCs (p < 0.001 and p < 0.0001, respectively). On the contrary, Fe and Al were elevated in frozen-thawed BEPCs (p < 0.0001 and p < 0.01, respectively) together with Ni (p < 0.0001). In addition, decreased Zn (p < 0.05) was observed in cryopreserved AT-MSCs. In conclusion, the ICP-OES technique might be used to analyze the basic elemental composition of animal cells in fresh or frozen-thawed conditions. Nevertheless, additional studies are needed to reveal the possible impact of cryopreservation on cell fate by changing the content of intracellular minerals.

Keywords: ICP-OES; cryopreservation; minerals; rabbit; stem cells.

Figure 1

Comparison of the macro and micromineral concentrations presented in the three rabbit fresh cell cultures (A,B). EPCs—rabbit endothelial progenitor cells derived from peripheral blood, BEPCs—rabbit endothelial progenitor cells derived from bone marrow, AT-MSCs—rabbit adipose-tissue derived mesenchymal stem cells. The data from three independent cell cultures for each cell line are expressed as the means ± SD; *—the difference is statistically significant at p < 0.05; **—the difference is statistically significant at p < 0.01; ***—the difference is statistically significant at p < 0.001; ****—the difference is statistically significant at p < 0.0001. Measurements for Cu (AT-MSCs), Co (EPCs and BEPCs), Mn (all cell lines), and Sr (BEPCs) were under the detection limits.

Figure 2

Effect of cryopreservation on the composition of macrominerals in the rabbit primary cell lines (A–C). EPCs—rabbit endothelial progenitor cells derived from peripheral blood, BEPCs—rabbit endothelial progenitor cells derived from bone marrow, AT-MSCs—rabbit adipose-tissue derived mesenchymal stem cells. The data from three independent cell cultures for each cell line are expressed as the means ± SD; *—the difference is statistically significant at p < 0.05; **—the difference is statistically significant at p < 0.01; ****—the difference is statistically significant at p < 0.0001.

Figure 3

Effect of cryopreservation on the composition of microminerals in the rabbit primary cell lines (A–C). EPCs—rabbit endothelial progenitor cells derived from peripheral blood, BEPCs—rabbit endothelial progenitor cells derived from bone marrow, AT-MSCs—rabbit adipose-tissue derived mesenchymal stem cells. The data from three independent cell cultures for each cell line are expressed as the means ± SD; *—the difference is statistically significant at p < 0.05; **—the difference is statistically significant at p < 0.01; ***—the difference is statistically significant at p < 0.001; ****—the difference is statistically significant at p < 0.0001. Measurements for Co (fresh and frozen–thawed EPCs (A) and AT-MSCs (C)), Mn (fresh and frozen–thawed EPCs (A), BEPCs (B) and AT-MSCs (C)), Cu (fresh AT-MSCs (C)), and Ni (frozen–thawed AT-MSCs (C)) were under detection limits.