Development of a GMP-Compliant Separation Method for Isolating Wharton's Jelly Derived Mesenchymal Stromal Cells from the Umbilical Cord

Abstract

Abstract Background Wharton's jelly derived mesenchymal stem cells (UC-MSCs) hold great therapeutic potential in regenerative medicine. However, GMP-compliant optimal methods for isolating UC-MSCs from UC tissue are still lacking. Additionally, there is a dearth of detailed research spanning from laboratory-scale to pilot-scale studies. Therefore, it is essential to establish standardized protocols that ensure the reproducibility and safety of UC-MSC manufacturing. Methods In this study, we aimed to explore and optimize parameters for the enzymatic digestion method used for isolating UC-MSCs. These parameters included enzyme concentrations, digestion times, seeding densities, and culture media. Additionally, we conducted a comparative analysis between the explant method and enzymatic digestion method. Subsequently, we evaluated the consecutive passaging stability of UC-MSCs, specifically up to passage 9, using the optimized enzymatic digestion method. Finally, we developed and scaled up manufacturing processes, starting from laboratory-scale flask-based production and progressing to pilot-scale cell factory-based production. Results The optimal parameters for the enzymatic digestion method were determined to be a concentration of 0.4 PZ U/mL Collagenase NB6 and a digestion time of 3 hours, resulting in a higher quantity of P0 UC-MSCs. Additionally, we observed a positive correlation between the initial cell seeding density and the number of P0 UC-MSCs. Evaluation of different concentrations of human platelet lysate (hPL) revealed that 2% and 5% concentrations resulted in similar levels of cell expansion, whereas a 10% concentration led to decreased cell expansion. Comparative analysis revealed that the enzymatic digestion method exhibited faster outgrowth of UC-MSCs compared to the explant method. However, after subsequent passages, there were no significant differences between the explant and enzymatic digestion methods in terms of cell proliferation, cell viability, and immunophenotype. Notably, consecutive passaging of UC-MSCs using the enzymatic digestion method demonstrated stability, with maintained cellular characteristics and functionality. Passages 2 to 5 exhibited higher viability and proliferation ability. Moreover, we successfully developed scalable manufacturing processes from the laboratory scale to the pilot scale, ensuring consistent production of high-quality UC-MSCs. Conclusion Our study provides valuable insights into the optimization of UC tissue processing protocols, the parameters for the enzymatic digestion method, and the comparative analysis of different isolation methods. We also demonstrated the stability of consecutive passaging using this method. Moreover, our scalable manufacturing processes enable large-scale production of high-quality UC-MSCs. These findings contribute to the advancement of UC-MSC-based therapies in regenerative medicine.