Elson, KM, Fox, N, Tipper, JL et al. (4 more authors) (2015) Non-destructive monitoring of viability in an ex vivo organ culture model of osteochondral tissue. eCells and Materials Journal, 29. 356 - 369. ISSN 1473-2262
Abstract
Organ culture is an increasingly important tool in research, with advantages over monolayer cell culture due to the inherent natural environment of tissues. Successful organ cultures must retain cell viability. The aim of this study was to produce viable and non-viable osteochondral organ cultures to assess the accumulation of soluble markers in the conditioned medium for predicting tissue viability. Porcine femoral osteochondral plugs were cultured for 20 days, with the addition on day 6, of Triton X-100 (to induce necrosis), camptothecin (to induce apoptosis) or no toxic additives. Tissue viability was assessed by the tissue destructive XTT (sodium 3'-[1-[(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene-sulfonic acid hydrate) assay method and LIVE/DEAD® staining of the cartilage at days 0, 6 and 20. Tissue structure was assessed by histological evaluation using haematoxylin & eosin and safranin O. Conditioned medium was assessed every 3-4 days for glucose depletion, and levels of lactate dehydrogenase (LDH), alkaline phosphatase (AP), glycosaminoglycans (GAGs), and matrix metalloproteinase (MMP)-2 and MMP-9. Necrotic cultures immediately showed a reduction in glucose consumption, and an immediate increase in LDH, GAG, MMP-2 and MMP-9 levels. Apoptotic cultures showed a delayed reduction in glucose consumption and delayed increase in LDH, a small rise in MMP-2 and MMP-9, but no significant effect on GAGs released into the conditioned medium. The data showed that tissue viability could be monitored by assessing the conditioned medium for the aforementioned markers, negating the need for tissue destructive assays. Physiologically relevant whole- or part-joint organ culture models, necessary for research and pre-clinical assessment of therapies, could be monitored this way, reducing the need to sacrifice tissues to determine viability, and hence reducing the sample numbers necessary.
Metadata
Item Type: | Article |
---|---|
Authors/Creators: |
|
Copyright, Publisher and Additional Information: | © 2015, Author(s).Reproduced in accordance with the publisher's self-archiving policy. With kind permission of full reproduction from eCM journal (www.ecmjournal.org). Founded by scientists for the benefit of Science rather than profit. |
Keywords: | Organ culture; apoptosis; necrosis; viability monitoring; glucose; lactate dehydrogenase; matrix metalloproteinase; glycosaminoglycar |
Dates: |
|
Institution: | The University of Leeds |
Academic Units: | The University of Leeds > Faculty of Biological Sciences (Leeds) The University of Leeds > Faculty of Engineering & Physical Sciences (Leeds) > School of Mechanical Engineering (Leeds) > Institute of Medical and Biological Engineering (iMBE) (Leeds) The University of Leeds > Faculty of Medicine and Health (Leeds) > School of Dentistry (Leeds) > Oral Biology (Leeds) |
Depositing User: | Symplectic Publications |
Date Deposited: | 25 Jun 2015 11:32 |
Last Modified: | 21 Feb 2024 14:45 |
Published Version: | http://www.ecmjournal.org/journal/papers/vol029/pd... |
Status: | Published |
Publisher: | AO Research Institute Davos |
Identification Number: | 10.22203/ecm.v029a27 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:87215 |