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scRNA-seq identified 4 major cell populations, namely corticotroph tumor (73.6%), stromal (11.2%), progenitor (8.3%), and immune cells (6.8%). Microarray analysis revealed striking changes in extracellular matrix, cell adhesion and motility-related genes concordant with loss of ACTH secretion during conventional 2D culture. Based on these findings, we subsequently defined a series of crucial culture nutrients and scaffold modifications that provided a more favorable trophic and structural environment that could maintain ACTH secretion