The Optimal Combination of Cartilage Source and Apparatus for Long‐TermIn VitroChondrocyte Viability Analysis

Abstract

Most studies of long-term chondrocytes survival were for tissue banks. They showed a gradual reduction in the viable chondrocytes percentage as a function of time and ambient temperature, but the samples were harvested under optimal conditions. The aim of our study was to determine the most reliable combination of cartilage source and assay for the in vitro postmortem chondrocyte viability analysis in the conditions that imitate a dead body. Osteochondral cylinders were procured from femoral condyles and talar trochleas of three male donors and stored in the cell culture media at 4 ± 2°C and 23 ± 2°C. The samples were analyzed by a cell viability analyzer and a confocal laser scanning microscope (CLSM) initially 24-36 h after death and then in 4-week intervals. The results reconfirmed the significant influence of time (p = 0.0002), but not of the temperature (p = 0.237). The largest reproducibility was presented for the knee joint and the CLSM.

Viability of Human Articular Chondrocytes Harvested Postmortem: Changes with Time and Temperature ofIn VitroCulture Conditions

“…The main focus of the study was to test the prolonged viability of the articular cartilage, a tissue which is well protected during the decomposition of the corpse, under controlled laboratory conditions. The peripheral articular cartilage was favored over other cartilaginous tissues of the head (auricle, nasal septum) , neck (thyroid cartilage, larynx, trachea, spine), or torso (ribs, bronchi, shoulders, hips, spine), which are in close contact to the commensal bacteria reservoir. To make a coherent study group, only male donors between 20 and 50 years of age with nondegenerated knee joint surfaces and no record or systemic diseases were included.…”

“…In the current study, we attempted to replicate the conditions in dead bodies but in controlled conditions (the media with nutrients that were diffused to the cells, the amount of media was limited, the media was not exchanged, oxygen was limited and was not added, waste materials were not removed). The ambient temperature was constant; the unpredictable influence of microorganisms was prevented by the addition of chosen antibiotics; other environmental effects were excluded .…”

“…The viability analysis was discontinued when three consecutive samples showed no viable cell. A person unrelated to the research manually counted the number of the viable and nonviable cells in each region of interest .…”

“…We initially designed an in vitro study in a controlled laboratory environment as there were no systematic data available from the literature. According to our previous pilot studies, we combined the most reliable source of cartilage samples (femoral part of the knee), in vitro cell storage media (DMEM/F12 with additives), and the apparatus for the evaluation of chondrocyte viability confocal laser scanning microscope (CLSM) of the Live/Dead stained samples . The aim of the current study was to determine the 2‐month in vitro viability curves at different temperatures of human articular chondrocytes harvested postmortem .…”

Estimation of Postmortem Interval from Cartilage Changes of Rabbit Auricle

“…Postmortem changes of cartilage have received very little research. It should be noted that most of studies used cartilage as a tool for estimation of postmortem interval focused only on the viability of isolated chondrocytes (Alibegović et al, 2011, Alibegović et al 2012, Alibegović et al 2014, or within cartilage (Drobnic et al,2005). Also, several methods were used for examination of viability of chondrocytes some of which were H&E (Rogers et al 2011), manual counting under a microscope, viability cell analyzer and the flow cytometer of isolated chondrocyte in cell culture media (Alibegović et al, 2011), and confocal laser microscope (Alibegović et al 2014).…”