Optimizing Cell Isolation for Adipose- and Synovium Derived Mesenchymal Stem Cells from Infrapatellar Fat Pad.

Background: The infrapatellar fat pad (IFP) is a rich source of mesenchymal stem cells (MSCs) with dual contributions from adipose and synovial tissues. The heterogeneity of IFP-derived MSCs and the lack of standardized isolation protocols, however, hinder consistent therapeutic outcomes. This study aimed to optimize collagenase-based isolation protocols for IFP-MSCs, with a focus on the effects of enzyme concentration and treatment duration on tissue digestion, cell origin, viability, and functional properties.

Methods: IFP tissues harvested from patients undergoing knee arthroscopy were enzymatically digested using various collagenase concentrations (0.1-2%) and incubation times (2-48 h). Histological, immunohistochemical, flow cytometric, and functional assays were performed to evaluate tissue degradation, surface marker expression, colony-forming ability, and trilineage differentiation.

Results: Milder digestion conditions (2 h, 0.2-0.4% collagenase) preferentially extracted synovial membrane MSCs (CD55⁺ cells) and supported higher CFUs and chondrogenic/osteogenic differentiation. In contrast, prolonged digestion (48 h) led to increased cell yields and adipogenic differentiation, but reduced cell viability and percentage of synovial marker expression.

Conclusion: In conclusion, enzymatic digestion parameters critically influence the cellular composition and regenerative potential of IFP-MSCs. Optimizing collagenase treatment conditions allows for a more selective, lineage-based MSC harvest, offering a practical strategy for tailored regenerative utilization of IFP-MSCs.