Enhancing Stem Cell Isolation from the Murine Small Intestine (2011)

Undergraduates: Anand Baxi, Richard J. von Furstenberg

Faculty Advisor: Susan Henning
Department: Chemistry

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A major challenge in the field of gastrointestinal biology is the isolation of intestinal stem cells as well as the generation of viable cell suspensions suitable for Florescence-activated cell sorting (FACS). We used cell surface antibodies to isolate a population of intestinal epithelial cells. In order to verify that our prep was epithelial-specific, we selected against CD31, an endothelial marker, and selected for EpCAM, an epithelial marker. Our results showed that the prep was over 97% epithelial. The antibody used to isolate stem cells was anti-CD24. The CD24lo subpopulation was 40-fold enriched for Lgr5 and 5-fold enriched for Bmi1, two known stem cell transcripts. In the initial preps, the viability of the sorted CD24+ population was not ideal. Thus, we explored ways to optimize the viability. The typical approach to isolate jejunal cells is to use EDTA to release epithelial sheets and dispase to dissociate into single cells. These steps require significant shaking. Studies have shown that air bubbles have a deleterious effect on cell viability. Surfactants make it thermodynamically unfavorable for air bubbles to interact with the cells, inhibiting resultant cell death. Of the three different surfactants studied PF68 was found to be the best. With the addition of PF68 to the solutions during shaking steps, we showed that viability increased from 71% to 83% directly after isolation and from 67% to 81% after 2 hrs at room temp, the average time necessary for FACS.