Cultivation of enteroids from fresh and cryopreserved bovine duodenal tissues

Summary: In case fresh tissues are not available for crypt isolation from the intestine immediately after tissue collection, developing a method for tissue cryopreservation would help enteroid formation, an important in vitro model. This technical note describes an established method for intestinal tissue cryopreservation and resuscitation for cattle, as well as bovine enteroid cultivation. Fetal bovine serum (90%) and dimethyl sulfoxide (10%) were used as a tissue cryopreservation solution, and tissues were cryopreserved until use. Crypts were isolated from fresh and cryopreserved tissues of cattle using 2 mM ethylenediaminetetraacetic acid/phosphate-buffered saline, and isolated crypts embedded in basement membrane extract were seeded in a 24-well plate and formed enteroids within 24 hours. After 7 days of cultivation, fragments derived from enteroids from both fresh and cryopreserved tissues were reseeded in a new 24-well plate and were able to be cultivated for 7 days. This tissue cryopreservation method enables researchers to establish stable bovine enteroids to investigate intestinal functions.