Abstract
Cell Sheet (CS) Engineering is a regenerative medicine strategy proposed for the treatment of injured or diseased organs and tissues. In fact, several clinical trials are underway using CS-based methodologies. However, the clinical application of such cell-based methodologies poses several challenges related with the preservation of CS structure and function from the fabrication site to the bedside. Pausing cells at hypothermic temperatures has been suggested as a valuable method for short-term cell preservation. In this study, we tested the efficiency of two preservation strategies, one using culture medium supplementation with Rokepie and the other using the preservation solution Hypothermosol, in preserving human adipose stromal/stem cells (hASC) CS-like confluent cultures at 4°C, during 3 and 7 days. Both preservation strategies demonstrated excellent ability to preserve cell function during the first 3 days in hypothermia, as demonstrated by metabolic activity results and assessment of extracellular matrix integrity and differentiation potential. At the end of the 7th day of hypothermic incubation, the decrease in cell metabolic activity was more evident for all conditions. Nonetheless, hASC incubated with Rokepie and Hypothermosol retained a higher metabolic activity and extracellular matrix integrity in comparison with unsupplemented cells. Differentiation results for the later time point showed that supplementation with both Rokepie and Hypothermosol rescued adipogenic differentiation potential but only Rokepie was able to preserve hASC osteogenic potential.
Highlights
According to the annual report of Organ Procurement and Transplantation Network/The Scientific Registry of Transplant Recipients, in 2017, 115,000 people in the USA alone were waiting for an organ transplant [1] and this number is increasing every year
In order to assess the impact of hypothermic storage with RP and HTS over cell number and metabolic activity, cells were placed at 4 ̊C for 3 and 7 days and allowed to recover for 24 hours at 37 ̊C after each time point
At day 7, both RP and HTS presented a metabolic activity of about 25% of the control 0h, significantly higher than the unsupplemented condition (10.16%) (Fig 2A). double strand DNA (dsDNA) quantification of confluent cultures revealed that when preservation solutions were used, cell content was maintained along the timepoints, while significant cell loss was found in the unsupplemented condition (Fig 2B)
Summary
According to the annual report of Organ Procurement and Transplantation Network/The Scientific Registry of Transplant Recipients, in 2017, 115,000 people in the USA alone were waiting for an organ transplant [1] and this number is increasing every year. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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