Generating an Organ Culture Model to Simulate Early-Stage Intervertebral Disc Disease

Abstract

Source: Saravi, B. et al., A Proinflammatory, Degenerative Organ Culture Model to Simulate Early-Stage Intervertebral Disc Disease. J. Vis. Exp. (2021).

This video demonstrates the development of a pro-inflammatory and degenerative organ culture model for early-stage intervertebral disc (IVD) disease using TNF-α. It outlines the procedure for injecting TNF-α into the intervertebral disc and explains the mechanisms through which TNF-α induces inflammation and degradation of the IVD.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. Intervertebral disc (IVD) culture and loading

1. Transfer the intervertebral discs (IVDs) to IVD chambers and add IVD culture medium (Dulbecco's Modified Eagle Medium (DMEM, 4.5 g/L high glucose DMEM for the physiological group and 2 g/L low glucose DMEM for the pathological group) + 1% Penicillin/Streptomycin + 2% fetal calf serum + 1% ITS (contains 5 µg/mL insulin, 6 µg/mL transferrin, and 5 ng/mL selenious acid) + 50 µg/mL ascorbate-2-phosphate + 1% non-essential amino acid + 50 µg/mL antimicrobial agent for primary cells) and place in an incubator at 37 °C, 85% humidity and 5% CO₂.
2. Culture the discs for 4 days within a bioreactor system according to experimental groups. In the pathologic group, maintain degenerative loading conditions at 0.32-0.5 MPa, 5 Hz for 2 h/day. In the physiological control group, use a loading protocol of 0.02-0.2 MPa, 0.2 Hz for 2 h/day.
   NOTE: Position the IVDs in chambers containing 5 mL of IVD medium during the loading procedures. The volume depends on the size of the bioreactor's loading chambers. Between the loading procedures, place the IVDs in six-well plates with 7 mL of IVD culture medium for free-swelling recovery.
3. For analyzing the changes in disc height during the experimental period, measure the disc height with a caliper after IVD dissection (baseline) and then daily after the free swelling period and after dynamic loading for the experimental duration.

2. Intradiscal tumor necrosis factor-alpha (TNF-α) injection

1. Directly after the first dynamic loading cycle on day 1, place the IVDs in a Petri dish in a vertical position and stabilize the IVDs with a tweezer.
2. Inject recombinant TNF-α (100 ng in 70 µL of phosphate-buffered saline (PBS) per IVD) with a 30-gauge insulin needle into the nucleus pulposus tissue of the pathological group. Inject slowly at a speed of approximately 70 µL in 1 min.
3. After injection, pull the syringe halfway back within the IVD and pull the syringe plunger to create a vacuum that prevents the injected solution from leaking back before removing the needle and syringe completely from the IVD.
   ​NOTE: Perform a pilot experiment by injecting PBS containing trypan blue dye to evaluate the distribution of the injected solution after loading and overnight culture.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ascorbate-2-phosphate	Sigma-Aldrich, St. Louis, USA	A8960
TNF-alpha, recombinant human protein	R&D systems, Minnesota, USA	210-TA-005
Bovine IL-8 Do.it-Yourself ELISA	Kingfisher Biotech, St. Paul, USA	DIY1028B-003
Corning ITS Premix	Corning Inc., New York, USA	354350
DMEM high glucose	Gibco by life technologies, Carlsbad, USA	10741574
DMEM low glucose	Gibco by life technologies, Carlsbad, USA	11564446
Ethanol for molecular biology	Sigma-Aldrich, St. Louis, USA	09-0851
Fetal Bovine Serum (FBS)	Gibco by life technologies, Carlsbad, USA	A4766801
Non-essential amino acid solution	Gibco by life technologies, Carlsbad, USA	11140050
Penicillin/Streptomycin(P/S)	gibco by life technologies, Carlsbad, USA	11548876
Phosphate Buffer Solution, tablet	Sigma-Aldrich, St. Louis, USA	P4417
Primocin	InvivoGen, Sandiego, USA	ant-pm-05