Chondrocytes isolation from hyaline cartilage by continuous monitoring method

Abstract

Background: Articular cartilage has poor regenerative capacities. Numerous cartilage repair techniques are known, including implantation of autologous chondrocytes. Material and methods: From 18 rabbits pieces of cartilage were harvested from femoral condyle. Minced cartilage was treated with 0.25% trypsin-EDTA. In the 1st group (n=9) the cartilage was digested with 0.6% collagenase in 15 ml tubes by shaking in incubator at 37°C, 5%CO2 . In the 2nd group (n=9) digestion was performed in 25cm2 cell culture flasks placed on the lateral side, monitoring the process under a microscope after 120 minutes. The isolated cells were cultured to a 80-90% confluence. The chondrocytes were identified using histochemical staining after culturing for 16 days in overconfluence. Results: Chondrocytes isolation in the 1st group lasted a fixed 360 minutes, in the 2nd group – 140±10 minutes. In the 1stgroup were isolated 9.2x104 ±3.1x104 chondrocytes with a viability of 85.36±16.41%, but in the 2nd group – 1.6x105 ±3.4x104 chondrocytes with a viability of 98.09±3.85%. The mean period of cell culture in the 1st group was 15±2 days, in the 2nd group – 11±3 days. In first passage of the 1st group were obtained – 1.2x106 ±4.3x105 chondrocytes and in the 2nd group – 2.92x106 ±3.6x105 chondrocytes. The secreted extracellular matrix by chondrocytes was stained specifically for cartilaginous tissue. Conclusions: The method used for chondrocytes isolation has a direct impact on the number of isolated cells, their viability, but also upon the culture period and the number of cells obtained during the first passage.

Human Chondrocytes, Metabolism of Articular Cartilage, and Strategies for Application to Tissue Engineering

“…Jeyakumar et al noted after two weeks the redifferentiation of chondrocytes under hypoxic conditions, yielding 4% pO 2 in a culture medium enriched with platelet-rich plasma (PRP). In the study, it was observed that PRP had a stimulatory effect on the gene expression of the markers COL2A1 and SOX9 [127][128][129]. Jahr et al applied 2.5% pO 2 in the cultivation of articular chondrocytes while proving the expression of the chondrogenic markers ACAN and COL2A1 and the reduction of expression of the dedifferentiation markers COL1A1 and COL3A1.…”