Abstract
Methods have been developed in this laboratory for clonal growth of chondrocytes taken directly from adult rabbits (1). Serum was found to be an absolute requirement for growth of these cells in current media (2). Preliminary results have indicated that the addition of fetuin or fetuin-like growth factors to the medium was not sufficient for promoting growth of these cartilage cells in culture. The fractionation techniques commonly used to purify serum growth factors were found inadequate for obtaining a growth-promoting factor for these primary cells.
A series of Diaflo membranes of calibrated pore sizes was used to fractionate fetal bovine serum and to characterize its growth-promoting activity.
Materials and methods. Diaflo ultrafiltration membranes (Amicon Corp.) with molecular cutoff values from 500 to 100,000 daltons were used with a Model 50 ultrafiltration cell (Amicon).
Ultrafiltrations were done in a cold room at 4° and all the membranes were rinsed with 50 ml of cold 10-3 M sodium acetate at pH 7.5 before being used for the experiment.
Fetal bovine serum (Flow Laboratories) was used for all experiments. For ultrafiltration, 10 ml of serum was diluted with 4 vol of cold acetate buffer and ultrafiltered until the original serum volume remained in the cell. The ultrafiltrate was lyophilized and the residue was taken up in 10 ml of distilled water. This solution and the concentrate that remained in the cell were sterilized by filtration through 0.22 μ Millipore filters prerinsed with distilled water (3).
The protein concentration in serum and in each serum fraction was determined by a modified Lowry Method (4).
Biological activity. Rabbit ear cartilage cells were prepared and grown in culture as described before (2) except that the incubation period was reduced to 7 days and the colonies stained with 0.1% crystal violet.
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