Generating Dorsal Root Ganglion Explant and Dissociated Cell Culture
Transcription
Start with dorsal root ganglion or DRG tissues in a serum-free medium. The DRG tissue contains sensory neurons and satellite glial cells embedded in the extracellular matrix, ECM.
Seed them onto a culture dish coated with a gelatinous protein mixture to enhance tissue adhesion.
Over time, glial cells release neurotrophic growth factors that allow neuronal extensions, establishing a DRG explant culture.
To develop a dissociated cell culture, take these DRG explants in a tube. Treat them with collagenase to degrade ECM and then with trypsin, which disrupts the cell connections, releasing neurons and glial cells.
Add a medium enriched with serum to stop the enzymatic reaction.
Pipette the contents repeatedly to create a single-cell suspension and filter it to remove debris.
Centrifuge this cell suspension and remove the enzyme-containing supernatant.
Resuspend the cells in a neurobasal medium and transfer them onto a laminin-coated plate.
The satellite glial cells and sensory neurons adhere to the laminin-coated plate, establishing a dissociated cell culture.
