Technique and method development for intervertebral disc (IVD) research

Abstract

Intervertebral disc (IVD) degeneration is one of the major causes of low back pain. The direct and indirect cost of managing back pain posts heavy socioeconomic burden to the society. Improved technologies and techniques together with well documented experiments will benefit the research field by saving effort in doing the optimization in every laboratory and avoiding experiment failure due to incomplete understanding of the procedures. These can accelerate scientific discovery, reduce the sacrifice of animals and enable a more effective use of funding. Nucleus pulposus (NP) is the central part of IVD. Differences in matrix compositions in human NP clinical samples demand different cell isolation protocols for optimal results but there is no clear guide about this to date. Sub-optimal protocols may result in low cell yield, limited reliability of results or even failure of experiments. We experimented different isolation protocols to study different parameters involved and suggested some rules for cell isolation in three main applications: RNA extraction for phenotyping, cell isolation for cell culture, and characterization by flow cytometry. In addition, instead of extracting RNA from isolated cells, extraction of RNA from tissues directly may avoid the change of RNA levels during the cell isolation process. However, extraction of RNA directly from human and large animal IVD tissue is technically challenging due to its tough nature, low cell-to-matrix ratio and high proteoglycan content. Thus we developed a method for RNA extraction from bovine disc tissues by integrating the use of cryosectioning, additional phase separation and high salt precipitation into conventional guanidinium thiocyanate based method. With this method, RNA could be extracted from the NP tissue directly but the concentration was low. A shift toward 270 nm was observed in its UV spectrum which was due to phenol contamination. This caused an overestimation of RNA concentration. Hence we developed a computational method based on UV spectra for correcting the overestimated concentrations of RNA contaminated with phenol. The accuracy of concentration increased substantially with the use of the correction formula. Mesenchymal stem cells (MSCs) have great potential in IVD engineering and hence we studied the isolation and culturing of MSCs from different sources. Effect of cell shape was reported for MSCs but not for NP cells. Micropatterning can be used to pattern cells into different shapes and arrangements. Therefore, we optimized the preparation of bovine NP cell micropatterns and also investigated the preparation of cell micropatterns for confocal microscopy. Besides, we developed methods to study the gene expression of cells on patterns by RT-qPCR with and without prior selection of cells of interest by laser capture microdissection (LCM). In short, different methods related to IVD research were developed and optimized. With improved methods together with a better understanding of the underlying rationale, researchers can save time and cost in their experiments and reduce experiment failure rate. This will help to accelerate researches. New methods also enable studies which were not feasible in the past.