A novel and efficient method for culturing mouse nucleus pulposus cells

Abstract

Background context: As degeneration of the nucleus pulposus (NP) is a major cause of intervertebral disc degeneration, research directed toward nucleus pulposus cells (NPCs) is drawing increased attention. However, caused by the difficulties associated with their harvest and culture, there are few reports describing cultivation methods for mouse NP cells (mNPCs).

Purpose: To establish efficient culture methods for mNPCs.

Study design: In vitro animal study.

Methods: After primary 3-dimensional (3D) gel culture of mNPCs and analysis of gene expression, cells digested from the gel were cultured in various bio-coated dishes with and without basic fibroblast growth factor (bFGF), and their growth kinetics and changes in gene expression profiles were evaluated. Next, the mNPCs obtained after sequential 3D gel and 2D culture were subjected to micromass culture and the effects of adding transforming growth factor-β3 (TGF-β3) on their gene expression profile and extracellular matrix (ECM) synthesis were evaluated.

Results: The cell morphology and gene expression pattern of mNPCs proliferated in primary 3D collagen gel culture resembled those of mNP. In contrast, mNPCs could not proliferate in conventional monolayer culture. Cell adhesion (colony number) and proliferation (colony size) were greater in fibronectin-coated dishes than in dishes with other bio-coatings. The addition of bFGF enhanced mNPCs proliferation, but the gene expression characteristics of mNPCs were lost as passage number increased. 2D culture with bFGF followed by micromass culture allowed for the recovery of the mNPC gene expression profile in primary 3D-gel culture, and TGF-β3 supplementation during micromass culture enhanced ECM synthesis.

Conclusions: We established novel culture methods for mNPCs. These methods will benefit basic cell-based and molecular research involving these cells.

Keywords: 3D collagen gel culture; Fibroblast growth factor; Fibronectin-coated dish; Micromass culture; Mouse nucleus pulposus cells; Transforming growth factor-β.