A Hybrid Hydrogel/Microsphere-Based Coculture System for Hematopoietic Stem Cell Maintenance

The ex vivo expansion of hematopoietic stem/progenitor cells derived from umbilical cord blood is limited by insufficient cell numbers and the loss of stemness. To address this problem, this study developed a microsphere-based dynamic coculture system. In this system, mononuclear cells or CD34+ cells were encapsulated within calcium alginate/gelatin hydrogel microbeads, while osteogenically induced MC3T3-E1 cells were immobilized on the surface of poly(lactic acid)/nanohydroxyapatite microspheres to establish a three-dimensional coculture environment. A systematic comparison was carried out among four culture modalities: static monoculture, static coculture, rotary cell culture system, and spinner flask. Results indicated that the rotary-based dynamic coculture system demonstrated superior performance, achieving a 6.56 ± 0.32-fold expansion of mononuclear cells, along with 7.85- and 7.53-fold increases in CD45+CD34+ and CD34+CD38– subpopulations, respectively. This group also had the lowest apoptosis rate (14.67% ± 2.87%) and the highest colony-forming capacity (445 ± 24 colonies). In the CD34+ cell culture, the rotary group further enhanced the expansion to 11.87 ± 0.74-fold, with a 15.72-fold increase in CD34+CD38– cells and 546 ± 39 colonies formed. Additionally, the secretion levels of ANGPT1 and Opn were significantly upregulated. These findings suggest that the microsphere-based dynamic coculture system offers a robust and scalable platform for the efficient ex vivo expansion of hematopoietic stem/progenitor cells and shows great potential for applications in hematopoietic tissue engineering.