*Purpose: Cold storage (CS) is the current gold standard preservation method for small bowel (SB) prior to transplantation. However, CS can cause epithelial detachment, sloughing, and crypt damage. Epithelial loss can lead to bacterial translocation and sepsis, thus, epithelial regeneration is critical to both allograft and patient survival. Intestinal epithelial stem cells (ISC) are the source of epithelial regeneration, however, the extent to which CS injures ISCs, ultimately impairing epithelial repair, is unclear. Currently, allograft health is monitored post-transplantation via histologic assessment of ileal biopsies. However, the ileum may not represent overall graft health as there is histologic evidence that the ileum may either be more resistant to CS injury or may have a greater capacity to repair than other SB segments. The impact of CS on ISCs from different SB segments is not well characterized. We hypothesized that CS would negatively impact ISC viability and proliferative potential, but that the ileum would be affected less than either the duodenum or jejunum.

*Methods: 9 porcine SB grafts were flushed with cold UW preservation solution and stored at 4ºC for 6h. 3-D ISC culture was used to determine the impact of CS on ISC viability and proliferative potential. Crypts isolated from duodenum, jejunum, and ileum segments obtained post-flushing (control, CO) and after 6h preservation were plated in culture. To assess ISC proliferative potential, spheroid area measurements were performed every 24h. To assess ISC viability, plating efficiencies were determined every 48h. Differences within and between groups were compared using a Kruskal-Wallis test and a Dunn’s multiple comparison post-test followed by a Mann-Whitney test. Significance was set at P<0.05.

*Results: CS jejunal spheroid area measurements were significantly smaller at all time points compared to CO tissues. When spheroid area measurements were compared between CS segments, ileal spheroids were significantly larger than duodenal or jejunal spheroids at all time points (P<0.0001). No significant differences were found when plating efficiencies were compared between CS and CO conditions for any segment.

*Conclusions: CS injury does not appear to cause a difference in ISC viability between SB segments, but the ISC proliferative potential is significantly greater in the ileum compared to the duodenum or jejunum. Our findings are important clinically, as ileal biopsies from transplanted SB serve as the marker of the health of the allograft based on histologic morphology. As a result, the ileum may not be the best marker of graft regenerative capacity, however the addition of future porcine CS procedure data is warranted before a final conclusion can be made.

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