Abstract
Cells isolated from intervertebral disc (IVD) tissues of human surgical samples are one of potential sources for the IVD cellular therapy. The purpose of this study was to develop a new non-enzymatic method, “tissue incubation”, for isolating human IVD cells. The IVD tissues of annulus fibrosus (AF) and nucleus pulposus (NP) were incubated separately in tissue culture flasks with culture medium. After 7–10 days incubation, cells were able to migrate out of IVD tissues and proliferate in vitro. After 3–4 weeks culture, expanded cells were harvested by trypsinization, and the remaining tissues were transferred to a new flask for another round of incubation. The molecular phenotype of IVD cells from juvenile and adult human samples was evaluated by both flow cytometry analysis and immunocytochemical staining for the expression of protein markers of NP cells (CD24, CD54, CD239, integrin α6 and laminin α5). Flow cytometry confirmed that both AF and NP cells of all ages positively expressed CD54 and integrin α6, with higher expression levels in NP cells than in AF cells for the juvenile group sample. However, CD24 expression was only found in juvenile NP cells, and not in AF or older disc cells. Similar expression patterns for NP markers were also confirmed by immunocytochemistry. In summary, this new non-enzymatic tissue incubation method for cell isolation preserves molecular phenotypic markers of NP cells and may provide a valuable cell source for the study of NP regeneration strategies.
Highlights
The intervertebral disc (IVD) is the largest avascular structure in the body and is composed of three morphologically distinct regions, the central nucleus pulposus (NP), the peripheral annulus fibrosus (AF), and the cartilaginous endplates
The IVD tissues of annulus fibrosus (AF) and nucleus pulposus (NP) were incubated separately in tissue culture flasks with culture medium
The molecular phenotype of IVD cells from juvenile and adult human samples was evaluated by both flow cytometry analysis and immunocytochemical staining for the expression of protein markers of NP cells (CD24, CD54, CD239, integrin a6 and laminin a5)
Summary
The intervertebral disc (IVD) is the largest avascular structure in the body and is composed of three morphologically distinct regions, the central nucleus pulposus (NP), the peripheral annulus fibrosus (AF), and the cartilaginous endplates. Cells isolated via enzymatic digestion may suffer damage to cell surface receptors immediately upon isolation (Gilchrist et al 2007), require longer expansion time to recover, and need multiple passages to achieve sufficient cell numbers, with increased passages and longer expansion resulting in cell dedifferentiation (Wang et al 2001). To overcome these disadvantages, we have developed a new nonenzymatic method, ‘‘tissue incubation’’, for isolating disc cells. Our findings suggest that this method is effective for isolating phenotypically
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