Isolation and Dissection Procedures for EGC Isolation by Human Mucosal Biopsies

This chapter provides a detailed guide for isolating and dissecting enteric glial cells (EGCs) from human mucosal biopsies. EGCs play crucial roles in gut homeostasis and neuroimmune communication, making their isolation a valuable endeavor in neurogastroenterology research. The method outlined here aims to enable competent scientists, even those unfamiliar with the technique, to successfully isolate EGCs at their first attempt. It outlines a systematic approach for the collection, digestion, mechanical dissection, and purification of cells from human mucosal biopsies, primarily focused on gastrointestinal tissue. During the first step, tissue collection, the collaboration with clinical teams ensures timely acquisition of biopsies with minimal trauma. Biopsies are then promptly transferred to a cold solution to maintain viability during transport. The subsequent enzymatic digestion involves immersion of biopsies in a solution containing collagenase and Dispase II, optimizing conditions for maximal cell yield. Gentle agitation aids enzyme penetration without causing cellular stress. Mechanical dissection follows, where tissues are minced into uniform fragments using sterile instruments, maintaining sterile conditions to prevent contamination. Gentle trituration further disperses cells without introducing air bubbles. Finally, cell purification involves filtration to remove debris, followed by centrifugation to pellet cells. Careful resuspension in an appropriate medium ensures viability for downstream applications. Overall, this method offers a meticulous approach to isolate viable cells from human mucosal biopsies for further analysis.