Relationship between oxygen partial pressure and inhibition of cell aggregation of human adipose tissue-derived mesenchymal stem cells stored in cell preservation solutions

Abstract

Introduction: This study investigated the storage conditions under which cell aggregation occurs and the conditions that inhibit cell aggregation when human adipose tissue-derived mesenchymal stem cells (hADSCs) are stored in lactated Ringer's solution (LR) supplemented with 3% trehalose and 5% dextran 40 (LR-3T-5D).

Methods: We first examined the effects of storage temperature and time on the aggregation and viability of hADSCs stored in LR and LR-3T-5D. The cells were stored at 5 °C or 25 °C for various times up to 24 h. We then evaluated the effects of storage volume (250-2,000 μL), cell density (2.5-20 × 10⁵ cells/mL), and nitrogen gas replacement on aggregation, oxygen partial pressure (pO₂), and viability of hADSCs stored for 24 h at 25 °C in LR-3T-5D.

Results: When stored in LR-3T-5D, viability did not change under either condition compared with pre-storage, but the cell aggregation rate increased significantly with storage at 25 °C for 24 h (p<0.001). In LR, the aggregation rate did not change under either condition, but cell viability decreased significantly after 24 h at both 5 °C and 25 °C (p < 0.05). The cell aggregation rates and pO₂ tended to decrease with increasing solution volume and cell density. Nitrogen gas replacement significantly decreased the cell aggregation rate and pO₂ (p < 0.05). However, there were no differences in viability among cells stored under conditions of different storage volumes, densities, and nitrogen gas replacement.

Conclusions: Aggregation of cells after storage at 25 °C in LR-3T-5D may be suppressed by increasing the storage volume and cell density as well as by incorporating nitrogen replacement, which lowers the pO₂ in the solution.

Keywords: Cell aggregation; Cell preservation solution; Human adipose tissue–derived mesenchymal stem cell; Oxygen partial pressure.

Fig. 1

Micrographs of human adipose tissue–derived stem cells (hADSCs) after storage at 5 °C 24 h and 25 °C for each hour in lactated Ringer’s solution (LR) and LR with 3% trehalose and 5% dextran 40 (LR-3T-5D) (magnification 100×). Black arrows indicate cell aggregates (≥5 cells) and white arrows indicate cell clusters of 4 ≤ cells. Inset shows a magnified image of the aggregated cells and cell clusters of 4 ≤ cells.

Fig. 2

Aggregation rate (A) and viability (B) of hADSCs after storage at 5 °C and 25 °C for various times in lactated Ringer’s solution (LR) and LR-3T-5D. Data are presented as the mean ± SD (n = 4). Statistical analysis was performed using two-tailed Dunnett’s test vs. before storage (Pre): ∗p < 0.05, ∗∗∗p < 0.001, and using two-tailed non-paired t-test: †p < 0.05, ††p < 0.01, †††p < 0.001.

Fig. 3

Aggregation rate (A), oxygen partial pressure (pO₂) in the preservation solution (B), and viability (C) of hADSCs after storage at 25 °C for 24 h in various volumes of LR-3T-5D. Data are presented as the mean ± SD (n = 8–14). Statistical analysis was performed using two-tailed Dunnett’s test vs. before storage (Pre): ∗∗p < 0.01, ∗∗∗p < 0.001, and vs. 250 μL: ††p < 0.01, †††p < 0.001.

Fig. 4

Aggregation rate (A), pO₂ in the preservation solution (B), and viability (C) of hADSCs after storage at various cell densities at 25 °C for 24 h in LR-3T-5D. Data are presented as the mean ± SD (n = 5–7). Statistical analysis was performed using two-tailed Dunnett’s test before storage (Pre): ∗∗p < 0.01, ∗∗∗p < 0.001, and vs. 2.5 × 10⁵ cells/mL: †p < 0.05, ††p < 0.01, †††p < 0.001.

Fig. 5

Aggregation rate (A), pO₂ in the preservation solution (B), and viability (C) of hADSCs after storage at 25 °C for 24 h in LR-3T-5D in tubes with or without (control) nitrogen gas in the head space. Data are presented as the mean ± SD (n = 5). Statistical analysis was performed using two-tailed non-paired t-test: ∗p < 0.05, ∗∗∗p < 0.001.