A Systematic Reevaluation of Enzymatic Isolation Methodology for Nucleus Pulposus Cells
Authors: Lee J. T. Y. , Leung V. Y. L. , Tam V. , Cheung K. M. C. 
Published: June 2012

Introduction The nucleus pulposus (NP) is the central part of IVD and understanding its function is essential for studying IVD degeneration. Enzymatic digestion by collagenase is used to release cells from NP tissues for cell phenotype characterization or further in vitro cell culture studies. For cell isolation from NP of different species, various protocols exist and vary greatly in terms of enzyme concentration, digestion duration, and type of precollagenase treatments such as digestion by pronase, which is a mixture of different proteases. In contrast to NP from other species, the cells in bovine or human mature NP have fewer cell-cell contacts and are surrounded by less gel-like matrix rich in proteoglycan and collagen. Thus we hypothesized that the pronase digestion step is not necessary to break the proteins which bind cells together in bovine or human NP. Bovine intervertebral disk (IVD) is an attractive candidate for studying IVD degeneration due to its anatomical and mechanical loading similarity with IVD in skeletally developed humans. And studies using bovine system can be more clinically relevant compared with other species. The avoidance of pronase treatment may better preserve the cell surface epitopes, which are critical for cell characterization recognizing surface protein markers. In this optimization study, we used bovine NP as a model to test different types of precollagenase treatments, different collagenase concentrations and different digestion durations to provide information about the optimal conditions for obtaining cells from bovine NP and human NP for different aims such as higher yield, higher viability, or reliable phenotyping. Materials and Methods Bovine NP tissue was isolated from bovine tail and digested by collagenase II with and without the prior digestion with pronase, trypsin, or dipase. The effect of collagenase concentration and digestion duration was studied by using two collagenase concentrations (0.05% vs 0.2%) and two incubation time points (4 to 8 hours vs. overnight). The number of extracted cells was estimated by counting with hematocytometer and the dead cells identified using Trypan blue stain. On the other hand, longer digestion duration and higher enzyme concentration would speed up the digestion but damages may be induced after prolonged digestion. Thus, the quality and phenotype of cells after prolonged digestion (with 0.5% collagenase for overnight digestion) plated on cell culture dishes was assessed using RT-qPCR after different cell passages. The mRNA levels of bovine NP cell markers (ACAN, COL2A1, CDH2, KRT18, KRT19) were monitored with GAPDH as the housekeeping gene and fresh bovine muscle as a control. Results Among the pre-collagenase enzyme treatments, pronase gives the highest cell yield. Longer duration of digestion or higher collagenase concentration without the use of pronase can achieve similar yield as with the pronase digestion step, indicating that the pronase digestion step may not be necessary. Overnight digestion may yield cells up to 7 times more than 4 hours of digestion. The majority of cells were viable and able to attach and grow on cell culture dishes though more dead cells were observed with overnight digestion and high enzyme concentration. High mRNA levels of NP markers were detected for the plated cells extracted with overnight digestion and high collagenase concentration (0.5%) compared with fresh muscle samples, indicating that the NP phenotype of cells was preserved. There is no significant difference in ACAN and COL2A1 mRNA expression in cells of the first three passages. On the other side, high quality of RNA can only be extracted from plated cells but not cells directly extracted out from the tissue after digestion with high enzyme concentration for overnight. This may be due to the unfavorable conditions for cells to synthesize mRNA during the collagen digestion, implying that different cell extraction protocols should be designed for cells extracted for different purposes. In short, pronase digestion may be omitted for isolation of cells from bovine NP tissue and this may better preserve the protein markers on the cell surface for characterization experiments. Overnight collagenase digestion gives higher cell yield and the majority of cells remain alive, although mRNA cannot be extracted immediately due to the harsh digestion conditions for the cells. Conclusion This study shows that higher cell yield can be obtained with longer digestion time and higher collagenase concentration though the cell viability may be sacrificed meanwhile. Pronase digestion may not be desirable for cell characterization using surface protein markers and shorter digestion duration seems preferable when high quality RNA is required for reliable RT-qPCR gene expression study. Due to the similarity of bovine NP and human NP, the systematic investigation of the digestion conditions using bovine NP in this study provides valuable information in extracting cells from both bovine NP and human NP for different purposes. This allows researchers to devise their extraction protocol based on a clear rationale, thus enabling more reliable results to be obtained in an efficient manner. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared