Harmonization and standardization of nucleus pulposus cell extraction and culture methods

Shaghayegh Basatvat, Frances C. Bach, Marcos N. Barcellona, Abbie L. Binch, Conor T. Buckley, Brian Bueno, Nadeen O. Chahine, Ana Chee, Laura B. Creemers, Stefan Dudli, Bailey Fearing, Stephen J. Ferguson, Jennifer Gansau, Benjamin Gantenbein, Rahul Gawri, Juliane D. Glaeser, Sibylle Grad, Julien Guerrero, Lisbet Haglund, Paula A. HernandezJudith A. Hoyland, Charles Huang, James C. Iatridis, Svenja Illien-Junger, Liufang Jing, Petra Kraus, Lisanne T. Laagland, Gernot Lang, Victor Leung, Zhen Li, Thomas Lufkin, Josette C. van Maanen, Emily E. McDonnell, Chris J. Panebianco, Steven M. Presciutti, Sanjna Rao, Stephen M. Richardson, Sarah Romereim, Tara C. Schmitz, Jordy Schol, Lori Setton, Dmitriy Sheyn, Joseph W. Snuggs, Y. Sun, Xiaohong Tan, Marianna A. Tryfonidou, Nam Vo, Dong Wang, Brandon Williams, Rebecca Williams, S. Tim Yoon, Christine L. Le Maitre

Research output: Contribution to journalArticlepeer-review

8 Scopus citations


Background: In vitro studies using nucleus pulposus (NP) cells are commonly used to investigate disc cell biology and pathogenesis, or to aid in the development of new therapies. However, lab-to-lab variability jeopardizes the much-needed progress in the field. Here, an international group of spine scientists collaborated to standardize extraction and expansion techniques for NP cells to reduce variability, improve comparability between labs and improve utilization of funding and resources. Methods: The most commonly applied methods for NP cell extraction, expansion, and re-differentiation were identified using a questionnaire to research groups worldwide. NP cell extraction methods from rat, rabbit, pig, dog, cow, and human NP tissue were experimentally assessed. Expansion and re-differentiation media and techniques were also investigated. Results: Recommended protocols are provided for extraction, expansion, and re-differentiation of NP cells from common species utilized for NP cell culture. Conclusions: This international, multilab and multispecies study identified cell extraction methods for greater cell yield and fewer gene expression changes by applying species-specific pronase usage, 60–100 U/ml collagenase for shorter durations. Recommendations for NP cell expansion, passage number, and many factors driving successful cell culture in different species are also addressed to support harmonization, rigor, and cross-lab comparisons on NP cells worldwide.

Original languageEnglish
Article numbere1238
JournalJOR Spine
Issue number1
StatePublished - Mar 2023


  • culture
  • harmonization
  • in vitro
  • intervertebral
  • nucleus pulposus
  • standardization


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