Effect of freezing on rabbit cultured chondrocytes

Abstract

This work evaluated the effect of freezing on chondrocytes maintained in culture, aiming the establishment of a cell bank for future application as heterologous implant. Chondrocytes extracted from joint cartilage of nine healthy New Zealand White rabbits were cultivated and frozen with the cryoprotector 5% dimethylsulfoxide for six months. Phenotypic and scanning electron microscopy analyses were carried out to identify morphological and functional differences between fresh and thawed cells. After enzymatic digestion, a total of 4.8x10 5 cells per rabbit were obtained. Fresh chondrocytes showed a high mitotic rate and abundant matrix was present up to 60 days of culture. Loss of phenotypic stability was notable in the thawed chondrocytes, with a low labeling of proteoglycans and weak immunostaining of type II collagen. The present study showed important loss of chondrocyte viability under the freezing conditions. For future in vivo studies of heterologous implant, these results suggests that a high number of cells should be implanted in the host site in order to achieve an adequate number of viable cells. Furthermore, the chondrocytes should be implanted after two weeks of culture, when the highest viability rate is found.

Platelet rich plasma associated with heterologous fresh and thawed chondrocytes on osteochondral lesions of rabbits

“…The technique used for cell culture was adapted from Filgueiras et al (2011). Cartilage fragments were deposited in sterile plastic tube containing 10mL of Ham's F12 culture medium (Ham's F12, Gibco-Invitrogen, USA) with addition of a 1% antibiotic-antimycotic solution (Penicillin G, streptomycin and anfotericin B; GibcoInvitrogen, USA) that was maintained under refrigeration until use.…”