Adipose-derived stem cells (ADSCs) and umbilical cord-derived mesenchymal stem cells (UCMSCs) were sourced from the Cellbank at the Stem Cell Institute, University of Science, Vietnam National University, Ho Chi Minh City (VNUHCM), Vietnam. ADSCs were thawed and cultured in ADSCCult I medium (Regenmedlab, Ho Chi Minh City, Vietnam), while UCMSCs were thawed and cultured in MSCult I medium (Regenmedlab, Ho Chi Minh City, Vietnam). Subculturing of these cells was performed using the enzyme Deattachment (Regenmedlab, Ho Chi Minh City, Vietnam) at a splitting ratio of 1:3.

ADSCs and UCMSCs were harvested and cryopreserved in MSCCryosave OTS medium, a DMSO-free cryopreservation medium (Regenmedlab, Ho Chi Minh City, Vietnam), at a concentration of 10⁷ cells/mL per cryovial. The cryovials were cooled at a rate of 1°C per minute in a freezing box and stored overnight at -86°C. All vials were subsequently transferred to liquid nitrogen tanks the following day.

The thawing process was conducted according to the manufacturer’s guidelines. Briefly, in step 1, the cryovials containing ADSCs and UCMSCs were transferred from the liquid nitrogen tank to a -86°C freezer overnight. In step 2, the vials were placed at room temperature to allow for natural thawing. The ice in the vials completely thawed within 5 to 10 minutes. Once the ice was completely dissolved, the cell suspension was mixed with Afterfreeze solution (Regenmedlab, HCMC, VN) in a 1:1 ratio (cell suspension: Afterfreeze solution). The thawed vials were then used in subsequent experiments.

A 30 mL thawed cell suspension, comprising 15 mL of MSCCryosave OTS and 15 mL of Afterfreeze from 15 vials, was administered into a 500 mL Lactated Ringer's solution bottle. The infusion tube was attached to this bottle, and the flow rate was adjusted to 25 drops per minute. Approximately 20-30 drops were collected every 30 minutes to assess cell viability (Figure 1). The experiments were conducted in triplicate.

Cell viability was assessed using a hemocytometer with trypan blue staining. To evaluate cell viability using Trypan Blue and a hemocytometer, harvested cells were first suspended in a liquid medium. The cell suspension was then mixed with an equal volume of 0.4% Trypan Blue solution. The hemocytometer was cleaned, and a coverslip was placed over it. The stained cell mixture was loaded into the chamber, allowing it to distribute by capillary action. Under low magnification on a microscope, cells were counted on the grid: clear cells were counted as viable, while blue-stained cells were considered non-viable. For accuracy, a minimum of 100 cells was counted. Viability was calculated by dividing the number of living cells by the total number of cells and adjusting for the dilution factor.

Each experiment was independently repeated three times. Results are expressed as the mean ± standard deviation (SD). Statistical significance was determined at p < 0.05. Data processing and plotting were performed using Prism software.

Figure 1

Figure 2

Figure 3

Figure 4

The data depicted in Figure 2 demonstrate that ADSCs suspended in Ringer lactate solution exhibit a progressive decline in viability as the infusion progresses. Specifically, after 90 minutes of infusion, the viability rate of the cells dropped to 80.41 ± 2.02%, from an initial rate of 92.95 ± 1.33% at 0 minutes (p < 0.05). Following the completion of a 530 mL infusion at 385 minutes, the viability rate further diminished to 48.22 ± 14.08%. Linear regression analysis reveals that the viability rate declines over time according to the equation y = -0.1168x + 91.506 (R² = 0.9945).

Similarly, UCMSC cells exhibit a declining survival rate over time (Figure 3). Initially, at 0 minutes, the cell survival rate was 94.44 ± 1.69%. This rate decreased to 81.12 ± 2.26% following 90 minutes of infusion and further declined to 59.39 ± 14.54% at 385 minutes (p < 0.05). After the infusion of 530 mL over 385 minutes, the survival rate remained quite low at 59.39 ± 14.54%. Linear regression analysis indicated that the cell survival rate decreased over time, following the equation y = -0.0902x + 90.585 (R² = 0.958).

The viability rate of MSCs, including ADSCs and UCMSCs, significantly increases when they are infused in a CellCarrier solution. For UCMSCs, there is no significant change in cell viability during the initial 30 minutes (89.77 ± 3.88 in Lactated Ringer vs. 93.87 ± 1.90 in CellCarrier, p > 0.05). However, at 60 minutes, the viability rates significantly differ between cells suspended in Lactated Ringer solution and those in CellCarrier solution (85.03 ± 3.22 in Lactated Ringer vs. 92.41 ± 1.78 in CellCarrier, p < 0.05). This difference becomes more pronounced from 90 minutes to 385 minutes (Figure 4A). At 385 minutes, the viability rate of UCMSCs in CellCarrier solution reaches 90.09 ± 0.44%, in contrast to only 49.32 ± 15.31% in Lactated Ringer solution (p < 0.01).

ADSCs exhibit a survival pattern similar to UCMSCs. As illustrated in Figure 4B, the survival rates of ADSCs are comparable in both Lactated Ringer and CellCarrier solutions during the initial 30 minutes (90.77 ± 4.08% in Lactated Ringer vs. 92.87 ± 1.80% in CellCarrier; p > 0.05). However, from 60 to 385 minutes, the survival rate in the CellCarrier solution is significantly higher than in the Lactated Ringer solution (p < 0.05). Specifically, at minute 60, the survival rate in the Lactated Ringer solution is 88.03 ± 4.12%, whereas it is 92.41 ± 1.58% in the CellCarrier solution (p < 0.05). By minute 385, the survival rate drops to 50.32 ± 13.31% in the Lactated Ringer solution, while it remains at 90.13 ± 0.24% in the CellCarrier solution (p < 0.01).