Abstract
Grossfeld(l) has shown that fibroblasts from different tissues cultivated in vitro are able to produce acid mucopolysaccharides (AMPS) and release them into the medium. AMPS belong to the group of hyaluronic acid and chondroitin sulphates A and C, are highly polymerized, and give positive mucin clot and hyaluronidase tests(2).
Since fibroblast-like cells are known to originate in cultures of buffy-coat and hemapoie-tic tissues(3,4), it seems of interest to study the AMPS production in human white blood cell cultures undergoing fibroblastic transformation.
Materials and methods. Peripheral human blood buffycoat was obtained by venipuncture and centrifugation at 600 × g with 0.1 mg/ml of heparin (Liquemine Roche) for each 5 ml of blood and phytoagglutinine (Difco), 0.25 ml for each 10 ml of blood. The first 1 or 2 ml extracted from the vein was discarded to avoid contamination with tissue cells. Buffycoats from 10 ml of blood were explanted in Earle's flasks with a layer of isologous plasma and 5 ml of liquid medium containing yeast extract Eagle mediumlactalbumin hydrolysate peptone (5) and 5% chicken embryo extract.
Turbidimetric tests of hyaluronidase according to Seastone and Kass(6) were performed as previously described(7) in samples of culture media taken every 4 days. The medium was centrifuged to eliminate cells and debris that might interfere with the turbidimetric tests. Incubated medium without explants was used as a blank. Determinations were also made on aliquots of homogenized cells before and after cultivation. The cells were homogenized in phosphate buffer pH 6.5 (0.1 M) with Potter Elvehjem homogenizer and centrifuged at 800 × g The supernatant was taken, for analysis.
Hyaluronidase from Benger Lab. Lt. was used (Hyalase Benger). It was found that 500 U Benger was the optimum necessary to reduce turbidity to 50%. Standard curves were made with pure hyaluronic acid prepared at Prof. Karl hIeyerls Laboratory.
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