Manipulation of Crypt Culture Conditions for Mouse Intestine Cell Fission (2013)
Undergraduate: Michele Bresler
Faculty Advisor: Susan Henning
Department: Biology
Background and Aim: The gastrointestinal epithelium is an organized tissue that constantly replenishes its lining with a high rate of turnover of every 3-5 days. This turnover is maintained by intestinal stem cells (ISCs) that exhibit self-renewal as well as multipotency. ISCs are located in intestinal crypts, which can be isolated and cultured to form enteroids which bud to form new crypt units. The objective of this project was to study various culture conditions for crypts, with the goal of identifying conditions in which crypts would be able to survive but would not undergo budding to form enteroids (ie minimal conditions for survival). This will make it easier for researchers to measure pro-proliferative effects of added factors in the future.
Methods: Normal crypt culture conditions contain Rspondin, Y27632, Noggin, EGF, and Wnt3a. All conditions used in this experiment contained Rspondin and Y27632 while Noggin, EGF, and Wnt3a were added variably. The percent of surviving crypts at 24h and at 7 days was measured, and the average bud number at 7 days was examined for each culture condition.
Results: It was determined that initial plating with R-spondin and Y27632, followed by a ¿rescue¿ at day 4 with Noggin and EGF was effective to allow crypt survival with minimal budding. Future studies will build on these conditions to assess whether bud number can be manipulated under different conditions to yield higher bud number.