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Non-destructive monitoring of viability in an ex vivo organ culture model of osteochondral tissue

机译：骨软骨组织离体器官培养模型中生存力的无损监测

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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 of Triton X-100 on day 6 (to induce necrosis), camptothecin (to induce apoptosis) or no toxic additives. Tissue viability was assessed by the tissue destructive XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide tetrazolium salt) 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.
机译：器官培养是研究中越来越重要的工具，由于组织固有的自然环境，它比单层细胞培养具有优势。成功的器官培养必须保留细胞活力。这项研究的目的是生产可行和不可行的骨软骨器官培养物，以评估可溶标志物在条件培养基中的积累，以预测组织的生存能力。猪股骨软骨栓塞培养20天，第6天添加Triton X-100（诱导坏死），喜树碱（诱导凋亡），或无毒性添加剂。通过组织破坏性XTT（2,3-双[2-甲氧基-4-硝基-5-磺基苯基] -2H-四唑鎓-5-羧基苯胺四唑鎓盐）测定方法和软骨的LIVE /DEAD®染色评估组织活力在第0、6和20天进行组织结构评估，方法是使用苏木精和曙红和番红花O进行组织学评估。每3-4天评估条件培养基的葡萄糖消耗以及乳酸脱氢酶（LDH），碱性磷酸酶（AP）的水平，糖胺聚糖（GAG）和基质金属蛋白酶（MMP）-2和MMP-9。坏死培养物立即显示葡萄糖消耗减少，而LDH，GAG，MMP-2和MMP-9水平立即增加。凋亡培养物显示葡萄糖消耗的延迟减少和LDH的延迟增加，MMP-2和MMP-9的少量增加，但对释放到条件培养基中的GAG却没有显着影响。数据表明，可以通过评估条件培养基中的上述标记物来监测组织活力，从而无需进行组织破坏性测定。对于研究和临床前评估疗法所必需的与生理相关的全关节或部分关节器官培养模型，可以通过这种方式进行监测，从而减少了牺牲组织以确定生存力的需要，从而减少了所需的样本数量。

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