title: Enzymatic isolation of chondrocytes from immature rabbit articular cartilage and maintenance of phenotypic expression in culture.
abstract: The studies included here identify factors affecting cartilage digestion by crude bacterial collagenase (cCGN) and describe a cartilage digestion medium that maximizes both tissue digestion rate and viable cell yield. The basal digestion medium contained 100 mM NaCl, 3 mM K2HPO4, 1 mM CaCl2, 1 mM MgSO4, 10 mM NaHCO3, 60 mM sorbitol, 5 mg/ml of dextrose, 1 mg/ml of albumin, and 2 mg/ml of cCGN in 25 mM HEPES at pH 7.2. Approximately 45% of articular cartilage tissue was digested in this basal medium in 6 h at 37 degrees C, yielding 6.8 x 10(6) viable cells per g tissue digested. The addition of 30 microM tosyllysylchloromethane (TLCM) increased the fraction of tissue digested in 6 h to 68% (p less than 0.05) and doubled viable cell yields to 13.6 x 10(6) per g tissue digested (p less than 0.05). Withholding Mg, decreasing NaCl to 70 mM, and adding 30 mM KCl increased fractional tissue digestion to 81% (p less than 0.01) and doubled viable cell yield yet again (to 29.9 x 10(6) viable cells per g tissue digested). Supplementation with TLCM increased the rate of cartilage digestion and the yield of viable cells regardless of cCGN source or lot. Additional trypsin (0.25%) inhibited tissue digestion and decreased cell yield; this effect was reversible with the addition of TLCM. The cartilage digestion medium developed in these studies (low Mg with added K and TLCM) was very effective in digesting articular, scapular, rib, and growth plate cartilage, as well as in yielding a large number of viable chondrocytes. These cells grew well in culture and maintained their chondrocytic characteristics, secreting predominantly type II collagen and large macromolecular forms of chondroitin sulfate-rich proteoglycans.
authors.affiliation: Department of Pharmacology, Northwestern University, Chicago, IL 60611.
authors.forename: M J
authors.initials: MJ
authors.lastname: Glade
authors.forename: Y S
authors.initials: YS
authors.lastname: Kanwar
authors.forename: T J
authors.initials: TJ
authors.lastname: Hefley
mesh_terms: D000818:Animals; D002358:Cartilage, Articular / Q000166:cytology* / Q000378:metabolism; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D003094:Collagen / Q000096:biosynthesis; D008297:Male; D003012:Microbial Collagenase; D010641:Phenotype; D011509:Proteoglycans / Q000096:biosynthesis; D011817:Rabbits; D013462:Sulfur Radioisotopes; D014316:Tritium
chemical_list: D011509:Proteoglycans; D013462:Sulfur Radioisotopes; D014316:Tritium; D003094:Collagen; D003012:Microbial Collagenase