An optimized step‐by‐step protocol for isolation of nucleus pulposus, annulus fibrosus, and end plate cells from the mouse intervertebral discs and subsequent preparation of high‐quality intact total RNA

Abstract

Intervertebral disc degeneration is the most significant, and least understood, cause of chronic back pain, affecting almost one in seven individuals at some point of time. Each intervertebral disc has three components; central nucleus pulposus (NP), concentric layers of annulus fibrosus (AF), and a pair of end plate (EP) that connects the disc to the vertebral bodies. Understanding the molecular and cellular basis of intervertebral disc growth, health, and aging will generate significant information for developing therapeutic approaches. Rapid and efficient preparations of homogeneous and pure cells are crucial for meaningful and rigorous downstream analysis at the cellular, molecular, and biochemical level. Cross-sample contamination may influence the interpretation of the results. In addition to altering gene expression, slow or delayed isolation procedures will also cause the degradation of cells and biomolecules that create a bias in the outcomes of the study. The mouse model system is extensively used to understand the intervertebral disc biology. Here we describe two protocols: (a) for efficient isolation of pure NP, AF, and EP cells from mouse lumbar intervertebral disc. We validated the purity of the NP and AF cells using Shh Cre/+ ; R26 mT/mG/+ dual-fluorescent reporter mice where all NP cells are GPF+ve, and by the sensitive approach of qPCR analysis using TaqMan probes for Shh, and Brachyury as NP-specific markers, Tenomodulin as AF-specific marker, and Osteocalcin as bonespecific marker. (b) For isolation of high-quality intact RNA with RIN of 9.3 to 10 from disc cells. These methods will be useful for the rigorous analysis of NP and AF cells, and improve our understanding of intervertebral disc biology.

Harmonization and standardization of nucleus pulposus cell extraction and culture methods

“… 39 Overall, we note that when working with NP tissues from smaller animals (mice, rats, and rabbits) it is important to obtain adequate tissue mass to effectively isolate enough cell numbers. While cell isolation from individual discs is possible for techniques such as RNA extraction 35 and single cell sequencing, 56 pooling together several discs from one animal increases feasibility. However, particularly for rodents and rabbits it may be technically difficult to obtain enough tissue from a single biological replicate for experiments requiring high cell numbers for forming 3D culture systems with cell yields per disc of 3–4 × 10 4 cells from rats and 15 × 10 4 cells from rabbit discs.…”

“…Mouse cell extraction methodology was not investigated further in this article as this was felt better placed in the companion article on NC extraction; furthermore, detailed protocols for mouse NP cells for downstream RNA analysis have recently been published. 35 Furthermore, given the similarity with rat tissues, and the larger NP tissue available from rat discs, rat tissue was selected for through testing within this study. Fourthly, culture of NP cells was investigated to determine the effect of basal media (within rabbit, cows, and humans), alternatives to fetal calf serum (FCS) (within dogs and humans) and differential substrates (within dog, pigs, and human), selected species were identified here, to represent key species utilized for such studies as initial indications for recommendations.…”

“…Extraction methodologies were investigated from NP tissue of humans, cows, dogs, pigs, rabbits, and rats, based on the protocols supplied and through expert review of methodologies, investigating enzyme types, concentrations and combinations, and digestion duration. Mouse cell extraction methodology was not investigated further in this article as this was felt better placed in the companion article on NC extraction; furthermore, detailed protocols for mouse NP cells for downstream RNA analysis have recently been published 35 . Furthermore, given the similarity with rat tissues, and the larger NP tissue available from rat discs, rat tissue was selected for through testing within this study.…”

Expansion of FGFR3-positive nucleus pulposus cells plays important roles in postnatal nucleus pulposus growth and regeneration

“…The surrounding ligament and muscle were removed from the vertebral column to expose the IVDs. After microdissection, lumbar (L1/2 ~ L5/6) and caudal (C6/7 ~ C10/11) NP tissues were collected and placed in separate RNA-free tubes on ice ( n = 4 ~ 5 mice per group) [ 28 , 29 ]. Total RNA was extracted using TRIzol reagent (Invitrogen, 15,596–026) according to the manufacturer’s instruction.…”

Improving reproducibility in spine research

“… Binch et al 1 shared the principles and protocols on immunostaining of the intervertebral disc, together with hints and tips on how to optimize any antibody for the intervertebral disc, with advice on what to avoid. Immunohistochemistry is a versatile technique enabling the detection and localization of specific protein expression, which is utilized in studies including developmental, pathogenesis, and investigating outcome measures for therapeutic approaches. Piprode et al 2 shared two protocols: one for isolation of each component of the murine intervertebral disc including nucleus pulposus, annulus fibrosus, and endplate cells, that can be used for further characterization by high‐throughput omics approaches including single cell RNAseq; and a protocol for isolation for high‐quality RNA along with pointers to simplify adaption. These methods will be useful for the rigorous analysis of IVD cells and will aid in the understanding of intervertebral disc biology.…”