Harmonization and standardization of nucleus pulposus cell extraction and culture methods
NP methodology paper; Tim Yoon, S; Le Maitre, Christine L
(2023) JOR Spine, volume 6, issue 1
(Article)
Abstract
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
... read more
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.
show less
Download/Full Text
Keywords: culture, harmonization, in vitro, intervertebral, nucleus pulposus, standardization
ISSN: 2572-1143
Publisher: Wiley-Blackwell Publishing Ltd
Note: Funding Information: AO Spine North America, ThéCell/Réseau de médecine régénérative de McGill (MRM) and The Montreal General Hospital Foundation Code Life; AOSPine International; Berta‐Ottenstein‐Programme for Advanced Clinician Scientists; Dutch Arthritis Foundation, Grant/Award Number: LLP22; H2020 European Institute of Innovation and Technology, Grant/Award Number: 825925; Irish Research Council for Science, Engineering and Technology, Grant/Award Number: GOIPG/2018/2448; Swiss National Science Foundation Project, Grant/Award Number: #310030E_192674/1; US National Institute of Health, Grant/Award Numbers: F32 AR070579, R01‐AR077678, R01‐AR080096, F31‐AR077385; Science Foundation Ireland Career Development Award, Grant/Award Number: 15/CDA/3476; National Science Foundation, Grant/Award Number: DGE‐2139839; Veterans Affairs Career Development Award, Office of Research and Development Biomedical Laboratory Research & Development, Grant/Award Number: IK2‐BX003845; Research Grant Council of Hong Kong, Grant/Award Number: GRF17121619; AOSpine, Grant/Award Number: AOSEAR2020‐02; NIH/NIAMS, Grant/Award Number: K01AR071512 Funding information Funding Information: This project has received funding from the European Union's Horizon 2020 Research and Innovation Programme under grant agreement no. 825925, named “iPSpine” (https://ipspine.eu, accessed June 19, 2022). MT, FB, LL have received funding from the Dutch Arthritis Foundation (LLP22). EMD, MB and CB were supported by the Irish Research Council (IRC) Government of Ireland Postgraduate Scholarship Scheme (GOIPG/2018/2448) and Science Foundation Ireland Career Development Award (15/CDA/3476). RG is supported by research grants from AO Spine North America, Thé Cell/Réseau de médecinerégénérative de McGill (MRM) and The Montreal General Hospital Foundation: Code Life. GL was supported by the Berta-Ottenstein-Programme for Advanced Clinician Scientists, Faculty of Medicine, University of Freiburg, Germany. BF is supported by NIH (F32 AR070579). LAS, XT and LJ were supported by funding from the US National Institutes of Health (R01-AR077678) and the National Science Foundation DGE-2139839. JI, JG, and CP were supported by funding from the US National Institutes of Health (R01-AR080096, and F31-AR077385). SMP was supported by a Veterans Affairs Career Development Award, Office of Research and Development Biomedical Laboratory Research & Development (IK2-BX003845). YS and VYL were supported by the Research Grant Council of Hong Kong (GRF17121619) and AOSpine (AOSEAR2020-02). BG acknowledges the Swiss National Science Foundation project #310030E_192674/1. DS was supported by NIH/NIAMS K01AR071512. ZL and SG would like to acknowledge AO Foundation; AOSpine International. The authors would like to thank the surgeons: Mr Ashley Cole, Mr Neil Chiverton, Mr Antony Michael, Mr Lee Breakwell, Mr Michael Athanassacopoulos, Mr Marcel Ivanov and Mr James Tomlinson from Northern General Hospital, Sheffield Teaching Hospitals NHS Trust for supply of human disc samples. The authors would like to thank the additional individuals for contributing cell culture methods: Sarah E. Gullbrand (Corporal Michael J. Crescenz VA Medical Center, Philadelphia, PA), Mauro Alini (AO Institute, Switzerland), Makarand Risbud (Thomas Jefferson University, Philadelphia), Keita Ito and Elias Salzer (TU/E), Grace O'Connell (University of California - Berkeley, Berkeley, CA), Kaito T and Kushioka J (Department of Orthopedic Surgery, Osaka University Graduate School of Medicine), Robert Bowles (University of Utah, USA), Oscar Alvarez-Garcia (Scripps Research Institute, La Jolla, CA), Rebecca Wachs (University of Nebraska-Lincoln), Shuichi Mizuno (Department of Orthopedic Surgery, Brigham and Women's Hospital and Harvard Medical School), Devina Purmessur (The Ohio State University, USA) and Chitra Dahia (Weill Cornell Medical College Medicine, Graduate School of Medical Sciences, Hospital for Special Surgery, New York, NY). Funding Information: This project has received funding from the European Union's Horizon 2020 Research and Innovation Programme under grant agreement no. 825925, named “iPSpine” ( https://ipspine.eu , accessed June 19, 2022). MT, FB, LL have received funding from the Dutch Arthritis Foundation (LLP22). EMD, MB and CB were supported by the Irish Research Council (IRC) Government of Ireland Postgraduate Scholarship Scheme (GOIPG/2018/2448) and Science Foundation Ireland Career Development Award (15/CDA/3476). RG is supported by research grants from AO Spine North America, Thé Cell/Réseau de médecinerégénérative de McGill (MRM) and The Montreal General Hospital Foundation: Code Life. GL was supported by the Berta‐Ottenstein‐Programme for Advanced Clinician Scientists, Faculty of Medicine, University of Freiburg, Germany. BF is supported by NIH (F32 AR070579). LAS, XT and LJ were supported by funding from the US National Institutes of Health (R01‐AR077678) and the National Science Foundation DGE‐2139839. JI, JG, and CP were supported by funding from the US National Institutes of Health (R01‐AR080096, and F31‐AR077385). SMP was supported by a Veterans Affairs Career Development Award, Office of Research and Development Biomedical Laboratory Research & Development (IK2‐BX003845). YS and VYL were supported by the Research Grant Council of Hong Kong (GRF17121619) and AOSpine (AOSEAR2020‐02). BG acknowledges the Swiss National Science Foundation project #310030E_192674/1. DS was supported by NIH/NIAMS K01AR071512. ZL and SG would like to acknowledge AO Foundation; AOSpine International. The authors would like to thank the surgeons: Mr Ashley Cole, Mr Neil Chiverton, Mr Antony Michael, Mr Lee Breakwell, Mr Michael Athanassacopoulos, Mr Marcel Ivanov and Mr James Tomlinson from Northern General Hospital, Sheffield Teaching Hospitals NHS Trust for supply of human disc samples. The authors would like to thank the additional individuals for contributing cell culture methods: Sarah E. Gullbrand (Corporal Michael J. Crescenz VA Medical Center, Philadelphia, PA), Mauro Alini (AO Institute, Switzerland), Makarand Risbud (Thomas Jefferson University, Philadelphia), Keita Ito and Elias Salzer (TU/E), Grace O'Connell (University of California ‐ Berkeley, Berkeley, CA), Kaito T and Kushioka J (Department of Orthopedic Surgery, Osaka University Graduate School of Medicine), Robert Bowles (University of Utah, USA), Oscar Alvarez‐Garcia (Scripps Research Institute, La Jolla, CA), Rebecca Wachs (University of Nebraska‐Lincoln), Shuichi Mizuno (Department of Orthopedic Surgery, Brigham and Women's Hospital and Harvard Medical School), Devina Purmessur (The Ohio State University, USA) and Chitra Dahia (Weill Cornell Medical College Medicine, Graduate School of Medical Sciences, Hospital for Special Surgery, New York, NY). Publisher Copyright: © 2023 The Authors. JOR Spine published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.
(Peer reviewed)
