How are Bovine Nucleus Pulposus Cell Populations Affected by Cell Isolation Methods?
Authors: Molinos M. M. , Almeida C. R. , Gonçalves R. M. , Barbosa M. A. 
Published: June 2012

Introduction

Strategies to promote intervertebral disk (IVD) regeneration have been hindered by the lack of knowledge of IVD fundamental cellular/molecular components. To date, many studies have been published culturing bovine IVD cells as a model to study IVD. However, the cellular mechanisms behind IVD de(re)generation remain largely unexplored. One of the key points to address is the characterization of the nucleus pulposus (NP) cell population(s), for which the isolation method is not consensual among the literature. These cells can be isolated using different enzymes and tissue digestion methods, but the effect of these parameters on the phenotype of cell population(s) has not been properly explored. This work aims to evaluate how the cell isolation method affects NP cell population(s).

Materials and Methods

NP cells were harvested from bovine caudal discs using six different digestion protocols - mechanical or enzymatic digestion, either with collagenase I, collagenase II, or both collagenase types (I + II). For mechanical tissue disruption, a gentleMACSDissociator was used. The dissociation time ranged from 4 to 19 hours. The effect of the dissociation method on NP cells immediately upon isolation was assessed by measuring cell yield (by counting cells), by viability (by flow cytometry), and by evaluating the cells morphology (by Imaging Flow Cytometry). Total 24 hours after tissue digestion, nonadherent cells were separated from the adherent fraction and the metabolic activity of both fractions was determined over time using resazurin assay. Prospective identification of different cell populations within bovine NP was performed using flow cytometry.

Results

Using the isolation methods described, the cell yield ranged from 4.9×105 to 1.1 × 106 cells per gram of tissue, with a cell viability between 59 and 82%. Comparing the different methods, collagenase type II digestion led to a higher cell yield (1.1 × 106 cells/g) and viability (82%). Cells isolated by the different protocols could be further characterized in terms of their morphology by measuring diverse parameters, such as cell length, elongation, shape, diameter, number of cell nucleus lobes, and number of vacuoles. Interestingly, three subpopulations of viable cells, with different sizes and auto-fluorescence, could be consistently identified by flow cytometry analysis, immediately upon isolation, without significant differences between the isolation protocols. Furthermore, two cell populations, adherent and nonadherent, could be isolated and cell metabolic activity suggests that it is possible to maintain both adherent and suspension cell populations in culture. Currently, the expression of specific cell markers for these populations is being evaluated.

Conclusion

Bovine NP cells were isolated using different separation methods. Digestion protocols using collagenase type II rendered higher cell yields and greater viability. Three different subpopulations of bovine NP cells were identified upon cell isolation. In monolayer culture two different cell subpopulations were separated and maintained in culture-adherent and nonadherent NP cells. Overall, this study demonstrates that the isolation method is crucial for the subsequent study of bovine NP cells. The identification of different NP cell populations and their dependence on the isolation method is particularly important when envisioning the identification of novel biological evidences that contribute to highlight IVD degeneration and its potential regenerative capacity. Acknowledgments This work was financed by FEDER funds through the Programa Operacional Factores de Competitividade - COMPETE, by portuguese funds through FCT - Fundação para a Ciência e a Tecnologia. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared