Dynamic Coculture Of Embedded UCB-CD34~+ Cell And Biomimetic Niche

Umbilical cord blood (UCB) as a valuable biological resource, has been applied to treat hematological malignancy, solid tumor, genetic disease and so on, with its transplatation advantages and abundant HSPCs. However, one UCB unit with lower HSPCs, limits its application in clinical. So it was an important strategy to expand UCB-HSPCs by engineered procedures in vitro. Currently, two main problems exist in traditional expansion methods:1) HSCs are inclined to differentiate earlier to lose sternness; 2) Allogenic stromal cells and exogenous factors are mixed with expanded HSCs to result in graft failure. While, it is found that in vivo bone marrow microenvironment allows HSCs to self-renew for a life span. Therefore, we first fabricate a biomimetic osteoblastic niche to provide an indirect support for HSPCs, then prepare an excellent gel-bead for embedding stromal cells to isolate allogenic stromal cells, finally expand embedded UCB-CD34+cell by biomimetic niche under dynamic condition.Fabrication of biomimetic osteoblastic niche:Firstly, HAp-CS/Gel scaffolds with varied ratio are prepared by freeze drying and chemical crosslink and it is found that HAp doping doesn’t destroy the morphology of scaffolds, which feature interconnected pores with size between 135 and 150μm. Then, the optimal HAp doping ratio of 100:20 is determined by measuring porosity, water uptake and contact angle. It is shown that the scaffold wih porosity bigger than 90% and water uptake folds of 19.1 becomes hydrophilic when its surface contact angle changes into 45.8° from 88.5°. In addition, by EDS and FITR analyses HAp uniformly distributes on the surface of scaffold wall with aggregates and particles, which provide enough sites for osteoblasts. Finally, osteoblasts are seeded into composite scaffold to fabricate biomimetic niche. Through fluorescence staining and SEM observation, it is found that cells can evenly attach on surface of scaffold and keep viable. Meanwhile, it demonstrates that osteoblasts can maintain their function and grow well on the scaffold by MTT and ALP tests.Preparation of CAG cell embedding matrix:Firstly, CAG gel-bead with "Core-shell" structure is prepared by simple droplet method and net-like channels of 18μm appear uniformly inner the gel-bead. Then, mechanical strength and diffusivity of gel-bead are measured and it shows that gel-beads can keep favorable strength in 2 weeks under dynamic stirring and semipermeable to hold back macromolecule protein. At last, osteoblasts are embedded in gel-beads to vertify that gel-beads are biocompatible and suitable to be as the embedded matrix.Dynamic coculture of embedded UCB-CD34+ cell and biomimetic niche:Firstly, UCB-CD34+ cells are isolated from UCB by MACs. It shows that isolated CD34+ cell keeps high viability of 93.2% with CD34 expression rate of 90.24%. After that, the CD34+ cells embedded by CAG gel-beads are co-cultured with biomimetic niche to explore the effects of dynamic condition and biomimetic niche on CD34+ cell, and two static control groups are set as contrasts. The expansion fold and viability of CD34+ cell are counted, which shows that CD34+ cells expand 24.2±2.3 folds and keep viability of 84.1% after dynamic coculture for 10 days, while in the static groups, especially the group without biomimetic niche support, cells expand slowly with a lower viability of 50%. CD34 expression rate is measured by flow cytometer, which reveals that CD34 expression rates all decrease while the rate in experimental group decrease slowly with a larger expression rate of 80.3% in 10th day. CFU-Cs are also counted to further investigate the potential maintenance of CD34+ cells, it indicates that the CFU-Cs in experimental group expand significantly with an expansion folds of 11.7±2.5 in contrast to the two static groups with far lower expansion folds. In a word, it is found that dynamic condition and biomimetic niche can jointly facilitate the expansion of CD34+ cell and preserve its viability and sternness.In summary, a novel expansion system including dynamic condition, biomimetic niche and embedded matrix, is fabricated to expand CD34+cell in vitro. This system enhances the transfer of nutrients and cytokines to provide timely stimuli to CD34+ cell and preserve its sternness. Based on the isolation by embedded matrix, it is promising to apply the expanded CD34+ cell into clinical.