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.

Involucrin, but not filaggrin and Kdap mRNA, expression is downregulated in 3-D cultures of intact rat hair bulbs after calcium stimulation

“…The deep layer containing hair bulbs was digested with 2 mg/ml collagenase and 1 mg/ml DNase (both from Sigma-Aldrich Chemie, Diesenhofen, Germany) dissolved in PBS at 37°C for one hour. Enzymatic solution contained 7 nM TLCK (a-tosyl-L-lysine chloromethyl ketone; Sigma-Aldrich), an inhibitor of clostripain present in collagenase preparations and harmful to cells [29]. After enzymatic treatment, the tissue was put through a 100-μm mesh sieve to remove larger fragments of dermis, washed three times with PBS solution and suspended in culture medium.…”