A step‐by‐step protocol for isolation of murine nucleus pulposus cells

Abstract

The intervertebral disc (IVD) is composed of three separate tissues with distinct origins and properties. Elucidating changes occurring in these tissues in response to injury or age is paramount to identify new therapies to better manage disc and spine degenerative conditions, including low back pain. Despite their small size and different mechanical load pattern compared to higher species, the use of mouse models represents a cost‐effective and powerful approach to better understand the formation, maintenance, and degeneration of the IVD. However, the isolation of the different compartments of the IVD is complicated by their diminutive size. Here, we describe a simple, step‐by‐step protocol for the isolation of the nucleus pulposus (NP) tissues that can then be processed for further analyses. Analysis from mouse NP tissues shows sufficient quantities of RNAs, purity of the NP fraction, and overall RNA quality for gene expression studies, and reveals no increase in expression of disc degeneration markers, including TNFa, IL1b, and Mmp1 up to 15 months of age in C57BL6 wildtype mice.

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.…”

Confocal scanning of intervertebral disc cells in3D: Inside alginate beads and in native microenvironment

“…Nucleus pulposus (NP) cells can be isolated from the intervertebral discs (IVD) of a variety species (eg, bovine, porcine, rodent, etc. ), and there are many different isolation protocols in literature 14,39 . While recommending an optimal protocol is beyond the scope of this study, we describe here the protocol used for isolating bovine NP cells.…”

“…), and there are many different isolation protocols in literature. 14,39 While recommending an optimal protocol is beyond the scope of this study, we describe here the protocol used for isolating bovine NP cells.…”

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

“…Several research groups studying disc biology use the mouse model system to elucidate changes in gene expression of NP cells 22‐31 . A few recent studies have also analyzed the gene expression of the mouse AF cells 24,28 .…”