20.500.12556/RUL-186567 High-content analysis of 3D chondrogenic pellets derived from primary cells In vitro 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-content analytical methods limit their translational use. Objectives: This study aimed to develop and optimize a high-content imaging workflow for quantitative evaluation of chondrogenesis in three-dimensional (3D) pellets 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 pellets were analyzed using high-content non-confocal imaging. Fluorescence intensities were adjusted to the pellet area to account for size-dependent effects. Results: Staining directly in imaging plates enabled streamlined high-content analysis. Controlled pepsin-mediated matrix digestion markedly enhanced antibody penetration, while excessive digestion compromised pellet 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 pellets. The optimized protocol enabled clear discrimination between chondrogenic pellets and controls, with approximately threefold higher type II collagen signal in chondrogenic samples. Conclusions: This study establishes a 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. 3D chondrogenesis high-content analysis immunofluorescence pellets primary MSCs type II collagen 3D hondrogeneza analiza visoke vsebnosti imunofluorescenca pelete primarne mezenhimske matične celice kolagen tipa II mezenhimske matične celice true false true Angleški jezik Slovenski jezik Članek v reviji 2026-09-03 07:55:22 2026-09-03 07:55:28 2026-09-04 04:30:43 0000-00-00 00:00:00 2026 0 0 22 str. iss. 7, art. 1496 Vol. 14 Jul. 2026 0000-00-00 Zaloznikova Objavljeno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 602.9 2227-9059 10.3390/biomedicines14071496 289574403 biomedicines-14-01496-v2.pdf biomedicines-14-01496-v2.pdf 1 2C356A7726E287FEDD421DD461A1C90E 1fdc54f325e84906a8075f90ea2c678dce0db5094e64687a4b7e6a1fed3d494c 3f6faaba-a75c-11f1-9b0d-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=245842 https://www.mdpi.com/2227-9059/14/7/1496 1 a38a3beb-a75b-11f1-9b0d-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=245841 Fakulteta za farmacijo Fakulteta za kemijo in kemijsko tehnologijo Medicinska fakulteta 0 0 0