title: In situ label-free cell viability assessment of nucleus pulposus tissue.
abstract: Regenerative medicine approaches aiming at treating degenerating intervertebral discs, a major cause of back pain, are increasingly tested in ex-vivo disc explant models mimicking in-vivo conditions. For assessing the efficacy of regenerative therapies, cell viability is commonly measured requiring specific labels to stain cells. Here, we demonstrate and evaluate how cellular auto-fluorescence can be utilized to non-invasively assess viability in disc tissue in-situ using label-free two-photon microscopy. Live and dead bovine disc cells (0% and 100% cell viability) from the nucleus pulposus were seeded into collagen gels and auto-fluorescence was characterized. Subsequently, nucleus pulposus explants were cultured for 6 days in media with different glucose supplementation (0, 0.25, 0.5, and 1 g/L) to induce different degrees of cell death. Then, samples were split and viability was assessed using label-free two-photon microscopy and conventional staining. Results show that live and dead nucleus pulposus cells systematically emit auto-fluorescent light with distinct characteristics. Cell viability values obtained with label-free microscopy did not significantly differ from those acquired with staining. In summary, monitoring auto-fluorescence facilitates accurate cell viability assessment in nucleus tissue requiring no additional dyes. Thus, this technique may be suitable for pre-clinical testing of regenerative therapies in nucleus pulposus cultures. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:545-550, 2014.
authors.affiliation: Orthopaedic Biomechanics, Department of Biomedical Engineering, Eindhoven University of Technology, PO Box 513, GEM-Z 4.115, 5600 MB Eindhoven, Eindhoven, The Netherlands.
authors.forename: Roman
authors.initials: R
authors.lastname: Dittmar
authors.forename: Bart G M
authors.initials: BG
authors.lastname: van Dijk
authors.forename: Marc A M J
authors.initials: MA
authors.lastname: van Zandvoort
authors.forename: Keita
authors.initials: K
authors.lastname: Ito
mesh_terms: D000818:Animals; D002417:Cattle; D002470:Cell Survival; D005453:Fluorescence; D007403:Intervertebral Disc / Q000473:pathology*; D055959:Intervertebral Disc Degeneration / Q000473:pathology; D018613:Microscopy, Confocal; D036641:Microscopy, Fluorescence, Multiphoton; D051381:Rats; D013050:Spectrometry, Fluorescence
keywords: auto-fluorescence; cell viability; intervertebral disc; label-free; regenerative medicine