Culture of equine intestinal epithelial stem cells after delayed tissue storage for future applications

BACKGROUND: Equine intestinal epithelial stem cells (ISCs) serve as potential targets to treat horses with severe intestinal injury. The ability to isolate and store ISCs from intestinal biopsies creates an opportunity for both in vitro experiments to study ISC dynamics in a variety of intestinal diseases, and, in the future, utilize these cells as a possible therapy. If biopsies could be successfully stored prior to processing for ISCs, this would increase the availability of sample repositories for future experimental and therapeutic use. However, delayed culture of equine ISCs following prolonged sample storage has not been described. The objective of this study was to describe the isolation and culture of equine ISCs following delayed tissue storage. Small intestinal full thickness biopsies were collected post euthanasia. Fresh tissue was immediately processed or stored at 4 °C for 24, 48 and 72 h (H) before processing. Intestinal stem cells (crypts) were dissociated and cultured. Size, growth efficiency and proliferation potential were compared between resultant enteroids (“mini-guts”) derived from each storage timepoint. In a separate study, growth efficiency of cryopreserved crypts was compared to cryopreserved enteroid fragments to investigate prolonged storage techniques. RESULTS: Intestinal crypts were successfully isolated and cultured from all timepoints. At 72H post initial collection, the intestine was friable with epithelial sloughing; resultant dissociation yielded more partial crypts. Enteroids grown from crypts isolated at 72H were smaller with less proliferative potential (bud units, (median 6.5, 3.75–14.25)) than control (median 25, 15–28, p < 0.0001). No statistical differences were noted from tissues stored for 24H compared to control. Following cryopreservation, growth efficiency improved when cells were stored as enteroid fragments (median 81.6%, 66.2–109) compared to crypts (median 21.2%, 20–21.5, p = 0.01). The main limitations included a small sample size and lack of additional functional assays on enteroids. CONCLUSIONS: Equine ISCs can be isolated and cultured after prolonged tissue storage. Resultant enteroids had minimal differences even after 24-48H of whole tissue storage. This suggests that ISCs could be isolated for several days from samples properly stored after procedures, including surgery or necropsy, and used to create ISC repositories for study or therapy of equine intestinal diseases.