고재윤 (가천대학교)

이후근 (가천대학교)

고재윤 (가천대학교)

목정훈 (가천대학교)

이후근 (가천대학교)

Recently, organoids have gained attention as three-dimensional cell models capable of recapitulating the structure and function of real organs. They are actively being studied as platforms for developing patient-specific regenerative therapies, including transplantation into damaged tissues to induce tissue regeneration. For the clinical and commercial application of such cell-based therapeutics, standardized manufacturing and quality evaluation systems are essential. However, objective criteria for assessing the quality and functionality of organoids remain insufficient. In this study, we aimed to characterize high-quality organoids and explore quantitative biomarkers for quality assessment by performing proteomic and metabolomic analyses on human-derived salivary gland organoids representing four cellular states: undifferentiated, ductal, serous, and mucous types. Proteomic analysis was conducted through LC-MS/MS following protein quantification using the BCA assay, in-solution digestion, and desalting. Metabolomic analysis was performed using LC-MS/MS after cell lysis with ammonium bicarbonate, methanol extraction, drying, and reconstitution. Data were analyzed using PCA and PLS-DA to visualize sample clustering, and differentially expressed proteins (DEPs) and metabolites (DEMs) were identified based on a VIP score ≥ 1.0. Proteomic comparison across differentiation states identified 703 (ductal vs. undifferentiated), 603 (serous vs. undifferentiated), and 649 (mucous vs. undifferentiated) DEPs, of which 324 were upregulated and 379 downregulated in ductal, 285 upregulated and 318 downregulated in serous, and 337 upregulated and 312 downregulated in mucous organoids. From these, the top 15 proteins based on VIP scores were selected as candidate signature markers. Metabolomic analysis identified 230 metabolites at MSI level 2, with 77, 82, and 88 DEMs detected in ductal, serous, and mucous groups, respectively. Among them, 56 metabolites were upregulated and 21 downregulated in ductal, 68 upregulated and 14 downregulated in serous, and 73 upregulated and 15 downregulated in mucous organoids. The top 15 metabolites were also selected as candidate signature markers. This analysis quantitatively compared proteomic and metabolomic profiles across differentiation types and confirmed that the resulting candidate markers are linked to the intrinsic functions of each cell lineage. These findings furnish baseline data that may support the future development of quality-assessment criteria for salivary-gland organoids