Optimization and comparison of different methods for assessing cell viability in intervertebral disc organ cultures.

Introduction: Whole-organ intervertebral disc (IVD) culture is widely used to study IVD degeneration and evaluate regenerative therapies. Spatial visualization of resident cell viability is essential, yet commonly used assays, such as Calcein AM/Ethidium Homodimer-1 (EthD-1), lactate dehydrogenase (LDH)/EthD-1, and MTT/DAPI, lack standardized protocols that facilitate implementation. Methods: In this study, we optimized and compared protocols for all three staining methods in bovine caudal IVDs and evaluated their strengths and limitations using appropriate positive and negative controls. Results: Selection of suitable controls was critical for reliable viability assessment. Calcein AM/EthD-1 provided a straightforward approach but required protocol modifications, including Collagenase P pre-treatment, to ensure adequate tissue penetration. This method also requires immediate processing and imaging after harvesting. In contrast, LDH/EthD-1 and MTT/DAPI allows for less time-sensitive workflows and enables repeated staining. However, LDH/EthD-1 is unsuitable for short-term experiments, as LDH signal persists for up to 36 h after cell death, except under extreme conditions such as repeated snap-freezing. MTT/DAPI proved more suitable for short-term applications. Discussion: These findings provide practical guidance for selecting and implementing viability assays in IVD culture, facilitating method choice based on specific experimental objectives and time constraints. Moreover, the insights gained may also be applicable to other dense tissues, such as tendon and cartilage.