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

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.

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.

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.

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.