Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture

Summary

This protocol illustrates a harvesting technique for coccygeal bovine intervertebral discs for organ culture for in vitro 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.

Protocol

1. Intervertebral Disc Harvesting

1. Whole length bovine tail is obtained from a local abattoir, if possible without skin since the presence of skin increases the chance of contamination (Fig 2).
2. Prepare large cutting board and prepare sterile work station and instruments on top of a cutting board (Fig. 2).
3. Prepare under the sterile laminar flow hood sterile gauze moistened with 0.9% sodium chloride containing 55mM sodium citrate and put into each well of the 6-well plate.
4. Prepare a basin and dilute 1:100 Betadine solution with tap water.
5. Immerse the whole bovine tail in a basin containing 1% Betadine solution for 5 min....

Discussion

The first step for successful organ culture is to make sure that the explant should not be contaminated. The tail should be skinned before you start with the procedure. Any animal hair brought into a sterile lab could be problematic in terms of contamination. The bovine tail should ideally be as fresh as possible (this affects initial cell viability). Furthermore, the betadine washing step is recommended to reduce the risk of contamination further. Instead of using a custom-made blade holder and a hammer to separate the .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fresh bovine intervertebral disc tissue from bovine tails, from local slaughter house (ideally within hours post-mortem and without skin).
Pulsavac Plus AC System	Zimmer inc., Switzerland	00-5150-486-01	Best performance with the hip-spray head and with AC power supply (the one with the 8 AA battery pack d–s also work but is less convenient)
High Capacity Fan Spray w/Splash Shield, 12.7cm length	Zimmer inc., Switzerland	00-5150-175-00	There are several spray heads available, we tested this one successfully
Scalpel blades #22 and #10	Swann-Morton	#10: 0201 #22: 0208
Scalpel blade holder # 3 and #4	Hausmann, Germany	#3: 06.103.00 #4: 06.104.00
Lutz industrial blade	Lutz, Germany	1022.0884
Phosphate buffered Saline (PBS)	Invitrogen	10010-023
Dulbecco’s Modified Eagle Medium (DMEM)	GIBCO, by Life Technologies	11960-044
Lactated Ringer’s solution (without glucose)	Bichsel, Switzerland	133 0002
6-well multi-well plate	Techno Plastic Products	92006
Betadine solution	Mundipharma, Switzerland	10055025
Surgical skin marker	Porex Surgical, Switzerland	9560
Large cutting board			Any brand is possible