Biological characteristics of adult degenerative nucleus pulposus cells in a three‐dimensional microcarrier stirring culture system

Abstract

A major problem in reconstructing degenerative intervertebral discs is to obtain sufficient nucleus pulposus (NP) seeding cells with normal physiologic functions. The current study adopted a three-dimensional microcarrier culture system for massive cell expansion and evaluated the biological characteristics and physiological functions of the propagated adult degenerative NP cells. Isolated adult NP cells were cultured in either microcarrier stirring culturing system or traditional monolayer cultivation. The growth characteristics, proliferation, extracellular matrix secretion, and apoptosis potential were examined to evaluate the different features of the two cultivation methods. Compared to the monolayer cultivation system, the adhesion time of NP cells in the three-dimensional microcarrier culture system appeared longer with relatively transient stable growth period. MTT and 3 H-TdR assays suggested significantly elevated proliferation and higher thymidine incorporation rates in cells from microcarrier system compare to cells in the monolayer system at the exponential growth phase (p < 0.05). Western blot data complimented the immunostaining results that the NP cells in the microcarrier system expressed significantly more protein levels of both type collagens at the exponential growth phase than that in the monolayer system (p < 0.05). Further, significantly more 35 S labeled proteoglycan incorporation was noticed in the cells on the microcarriers at both the stable growth and the exponential growth phases (p < 0.05 and p < 0.01). In conclusion, the three-dimensional microcarrier stirring culture system provides a means of fast and massive propagation of NP seeding cells which maintain their normal physiological characteristics and functions. ß

Peptide-Grafted Microspheres for Mesenchymal Stem Cell Sorting and Expansion by Selective Adhesion

“…Furthermore, we found that the number of MSCs captured on E7/PEG/pDA/Fe 3 O 4 -OA-PLGA under dynamic conditions was statistically significantly lower than the number of MSCs captured under static conditions ( Supplementary Table S1 ). Consistent with the preceding results, it is possible that shear forces affected early cell adhesion ( Ning et al, 2013 ), and such shear forces might, to some extent, also mimic the actual stress environment in vivo ( Zhang et al, 2010 ).…”

“…The total number of adherent cells on the surface of each microsphere decreased compared with that in static culture. Presumably, shear forces in the system may cause adhesion to take longer to achieve ( Ning et al, 2013 ). After modification with dopamine, the number of cells on the pDA/Fe 3 O 4 -OA-PLGA microspheres increased (versus the Fe 3 O 4 -OA-PLGA microspheres group).…”

In Situ Precipitation of Cluster and Acicular Hydroxyapatite onto Porous Poly(γ-benzyl-l-glutamate) Microcarriers for Bone Tissue Engineering

“…At 1, 4, and 7 days, the cell/microcarrier constructs were rinsed with PBS, stained using Calcein-AM/PI Double Staining Kit, and then observed under confocal laser microscopy (Nikon Y-FL, Japan). Cell proliferation was also detected by MTT assay, as previously reported …”

“…Cell proliferation was also detected by MTT assay, as previously reported. 47 After 12 h of incubation, the cell/microcarrier constructs were fixed with 4% paraformaldehyde solution. The nucleus and cytoskeleton were fluorescently stained with Hoechst 33258 solution and TPITC Phalloidin solution.…”