The application of patient-derived organoids (PDOs) in prostate cancer (PCa) research has been hampered by poor take rates and benign overgrowth. We highlight the limitations of existing culture conditions and identify extracellular matrix composition as a determinant of organoid outcome. Single-cell RNA sequencing reveals that Matrigel-free PDOs exhibit cellular heterogeneity, preserve patient-specific PCa cells with active androgen receptor signaling, and enrich in intermediate cells. In contrast, Matrigel fails to maintain primary PCa cells and produces in vitro basal-like features divergent from patient samples. Furthermore, we redefine cell-type signatures, identify biomarkers discriminating tumor versus other cell types, and show that expression of laminin-binding integrins is a hallmark of Matrigel-derived organoids. Finally, integrating previously published datasets with our data, we generate a prostate PDO single-cell atlas (PPScA), which captures a spectrum of cellular identities while revealing pathways altered in vitro. Our study provides methodological improvements for short-term culture and cellular biology insights.