20.500.12556/RUL-182207 High content analysis of 3D chondrogenic spheroids derived from primary cells in vitro research data underlying the article Background: Primary cells derived from connective tissues contain mesenchymal stem/stromal cell (MSC)–like progenitors with chondrogenic potential relevant for cartilage repair. However, donor‑ and tissue‑specific variability and the lack of robust, high‑throughput analytical methods limit their translational use. Objectives: This study aimed to develop and optimize a fast, reproducible high‑content imaging workflow for quantitative evaluation of chondrogenesis in three‑dimensional (3D) spheroids derived from primary cells. Methods: Primary human cells isolated from cartilage were chondrogenically differentiated in vitro. A systematic optimization of immunofluorescence staining parameters was performed, including staining platform, enzymatic matrix digestion, non‑specific site blocking, membrane permeabilization, and nuclear counterstaining. Type II collagen was detected using an Alexa Fluor 488–conjugated antibody, and spheroids were analyzed using high‑content non-confocal imaging. Fluorescence intensities were normalized to spheroid area to account for size‑dependent effects. Results: Staining directly in imaging plates enabled streamlined high‑content analysis. Controlled pep-sin‑mediated matrix digestion markedly enhanced antibody penetration, while excessive digestion compromised spheroid integrity. Extended bovine serum albumin blocking improved type II collagen signal intensity and homogeneity. Triton X‑100 permeabilization increased detection sensitivity but occasionally induced structural disruption in weakly organized control spheroids. The optimized protocol enabled clear discrimination be-tween chondrogenic spheroids and controls, with approximately threefold higher type II collagen signal in chondrogenic samples. Conclusions: This study establishes a stand-ardized, high‑content imaging–based workflow for quantitative assessment of 3D chondrogenesis from primary cells. The approach provides a rapid, scalable platform with direct relevance for in vitro screening, potency testing, and quality control in cartilage‑oriented advanced therapy development. primary progenitor cells chondrogenesis 3D spheroids high content analysis type II collagen immunofluorescence true false false Angleški jezik Slovenski jezik Neznano 2026-04-30 14:35:17 2026-05-18 22:51:58 2026-05-19 04:36:13 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 2026-10-01 README.docx README.docx README 1 771548ECD611AF59564192D229331038 8b8873bc645b962d2b03f2393c6c69217ced6205f24c123e8c2c0d853860bf55 17fb579d-4491-11f1-b0ab-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=233226 Raw_data-Control1.txt Raw_data-Control1.txt Raw data Control1 1 B33EF4DE0FBB636E6C7D8BFAF65AD2F1 cd9e09265e2f22abbedca9c80967b829ac17a553464358495012368c6e227368 129e5003-4491-11f1-b0ab-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=233216 Raw_data-Sample3.txt Raw_data-Sample3.txt Raw data Sample3 1 3DC27B632E68553B9DA81A3D991A7C1C b5517c896f20a926de85e0e67aa6103e8e57b93d47da53ecabb35a4c0bcfdcdb 16c96686-4491-11f1-b0ab-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=233217 Raw_data-Sample6.txt Raw_data-Sample6.txt Raw data Sample6 1 F22AC4750FB0F26DACCD0CF4D782CEA2 6fafc4c72a9e4a721170f1aff57a0560d67494a4cfb4aac60bacb1908f5ebf4c 16e18468-4491-11f1-b0ab-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=233218 Raw_data-Sample8.txt Raw_data-Sample8.txt Raw data Sample8 1 4326D2231265B211D097CC60F576E9D4 8be1040e027efbfab5fa0dfec3b29f99b2b3b0b3ace4d7648cf977cb0c892fc1 16f96da2-4491-11f1-b0ab-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=233219 Fakulteta za farmacijo Meritve in testi 0 0 0