title: Preparation of intact bovine tail intervertebral discs for organ culture.
abstract: The intervertebral disc (IVD) is the joint of the spine connecting  vertebra to vertebra. It functions to transmit loading of the spine and give  flexibility to the spine. It composes of three compartments: the innermost  nucleus pulposus (NP) encompassing by the annulus fibrosus (AF), and two cartilaginous  endplates connecting the NP and AF to the vertebral body on both sides. Discogenic  pain possibly caused by degenerative intervertebral disc disease (DDD) and disc  herniations has been identified as a major problem in our modern society. To  study possible mechanisms of IVD degeneration, in vitro organ culture  systems with live disc cells are highly appealing. The in vitro culture of intact  bovine coccygeal IVDs has advanced to a relevant model system, which allows the  study of mechano-biological aspects in a well-controlled physiological and  mechanical environment. Bovine tail IVDs can be obtained relatively easy in  higher numbers and are very similar to the human lumbar IVDs with respect to cell  density, cell population and dimensions. However, previous bovine caudal IVD  harvesting techniques retaining cartilaginous endplates and bony endplates  failed after 1-2 days of culture since the nutrition pathways were obviously  blocked by clotted blood. IVDs are the biggest avascular organs, thus, the  nutrients to the cells in the NP are solely dependent on diffusion via the  capillary buds from the adjacent vertebral body. Presence of bone debris and  clotted blood on the endplate surfaces can hinder nutrient diffusion into the  center of the disc and compromise cell viability. Our group established a relatively quick  protocol to "crack"-out the IVDs from the tail with a low risk for  contamination. We are able to permeabilize the freshly-cut bony endplate surfaces  by using a surgical jet lavage system, which removes the blood clots and  cutting debris and very efficiently reopens the nutrition diffusion pathway to  the center of the IVD. The presence of growth plates on both sides of the  vertebral bone has to be avoided and to be removed prior to culture. In this  video, we outline the crucial steps during preparation and demonstrate the key  to a successful organ culture maintaining high cell viability for 14 days under  free swelling culture. The culture time could be extended when appropriate  mechanical environment can be maintained by using mechanical loading bioreactor.  The technique demonstrated here can be extended to other animal species such as  porcine, ovine and leporine caudal and lumbar IVD isolation.
authors.affiliation: ARTORG Center for Biomedical Engineering, University of Bern, Switzerland.
authors.forename: Samantha C W
authors.initials: SC
authors.lastname: Chan
authors.forename: Benjamin
authors.initials: B
authors.lastname: Gantenbein-Ritter
mesh_terms: D000818:Animals; D002417:Cattle; D007403:Intervertebral Disc / Q000033:anatomy & histology / Q000254:growth & development*; D009924:Organ Culture Techniques / Q000379:methods*; D013623:Tail / Q000033:anatomy & histology