Comparative Analysis of Decellularization Protocols for Human Placenta: Balancing Cell Removal with Extracellular Matrix Preservation for Hydrogel Applications.

PURPOSE: The extracellular matrix (ECM) is crucial for tissue structure and cell behavior, positioning decellularized ECM (dECM) as a valuable biomaterial for regenerative medicine. The human placenta, abundant and bioactive, is an ideal ECM source. This study optimized decellularization protocols for human placental tissue to balance cellular removal with ECM integrity.

METHODS

Four protocols (P1-P4) were compared, using combinations of ionic/non-ionic detergents (SDS, Triton X-100), enzymatic agents (trypsin, DNase), and sterilizing treatments. Lyophilized dECM was processed into injectable hydrogels, characterized for mechanical, rheological, and degradation properties.

RESULTS

DNase-treated protocols reduced DNA to < 50 ng/mg dry weight, confirming effective cell removal, but ECM retention varied. P2, containing the highest concentrations of SDS among our tested protocols, results in depletion of glycosaminoglycans (GAGs), loss of fibronectin, and disruption of collagen organization. Milder protocols regarding SDS concentration, including P1 and P3, preserved GAGs and ECM proteins (collagen IV, fibronectin, laminin), as shown by immunofluorescence and histology. All protocols successfully formed stable hydrogels, but mechanical stiffness and viscoelastic properties differed. The high-SDS protocol without DNase treatment (P2-) exhibited the lowest storage modulus (G':13.7 ± 2.9 Pa), likely due to excessive ECM disruption. In contrast, the NaOH protocol (P3) with a lower SDS concentration than P2 showed the most consistent performance, with comparable G' values in DNase-treated (53.0 ± 20.0 Pa) and untreated (49.3 ± 3.3 Pa) hydrogels.

CONCLUSION

Detergent choice and DNase treatment influence ECM retention and hydrogel functionality. The NaOH protocol with DNase treatment (P3+) offers superior ECM preservation for placenta-derived dECM scaffolds, laying a foundation for regenerative medicine applications.