JACC are atelocollagen-associated autologous chondrocytes that form a cartilage-like tissue when cultured in a three-dimensional environment, using atelocollagen gel.
The cartilage biopsy sampes of patients (300-500 mg) are harvested, minced and washed three times in sterile sodium chloride (0.9%) supplemented with antibiotics (tobramycin 0.1mg/ml). Chondrocytes are isolated by incubation with trypsin (0.25%) in sterile saline, at 37°C for 30 minutes and then with collagenase (0.25%, 247g/mg) in Ham’s F-12 medium supplemented with patient serum (15%), HEPES buffer (10 mmol/l), gentamicin sulphate (50 g/ml), and amphotericin B (10 g/ml), for 4-6 hours. The isolated cells are collected by centrifugation (1500 rpm) and washed three times with the culture medium.
Two methods of culture are described:
Eight volumes of atelocollagen solution (3% type-I collagen) are added to one volume of concentrated Ham’s F-12 (10x) and one volume of NaOH (0.05N) with NaHCO2(2.2%) and 200 mM HEPES, with gentle agitation at 0°C. The cells are mixed thoroughly in the collagen medium mixture. The quantity of mixture required for implantation is determined by the size of the cartilage defect according to the following formula: defect area x 0.3 ml (final cell density = 2 × 106 cells/mL, 1.33% collagen). The mixture is then placed in a 60 mm diameter culture dish and completely gelated, by incubation at 37°C for 10 minutes, before being overlaid with 6 ml of the culture medium. Cell cultures are incubated in 5% carbon dioxide and 95% air at 37°C and fed with fresh medium containing L-ascorbic acid (50 g/ml), every two days.
The chondrocytes are suspended in Dulbecco’s modified medium (DMEM) supplemented with fetal bovine serum (10% FBS) and HEPES (20 mM). Four volumes of atelocollagen solution (3% type I collagen) are then added to one volume of cell suspension and mixed thoroughly. The mixture is placed onto culture dishes and gelated completely by incubation at 37°C. After 1 h, culture medium or DMEM supplemented with FBS (10%), and L-ascorbic acid phosphate magnesium salt (50 μg/ml), gentamicin sulfate (50 μg/ml), amphotericin B (0.25 μg/ml), and HEPES are added to the culture dishes. Then, the tissue-engineered cartilage is incubated in an atmosphere of 5% carbon dioxide and 95% air at 37°C. The culture medium is changed every 3–4 days.
EMBRYONIC DEVELOPMENT & STEM CELL COMPENDIUM
