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 aci....

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