Amniotic fluid cell culture. I. Experimental design for evaluating cell culture variables: determination of optimal fetal calf serum concentration.

Abstract

Extract: We have developed a randomized, multivariable block design for quantitative evaluation of conditions for amniotic fluid (AF) cell attachment to culture surface and subsequent growth rate. We report the use of this method to determine optimal fetal calf serum (FCS) concentration in the culture medium. The AF cell growth, in the form of discrete colonies, was scored by counting the number of colonies and measuring their diameters. A two-way analysis of variance of the preliminary experiment data (5–30% PCS) demonstrated a significant difference among AF cells grown at different percentages of FCS (P < 0.0005). From a studentized range analysis we concluded that 5% FCS is inferior to higher percentages. No difference was demonstrated among 15-30% FCS. Based on these data, a supplementary experiment was planned to compare 10 and 15% FCS. The 15% FCS yielded at least 0.31 colonies/ml AF more than did 10% FCS. We conclude that 15% FCS is sufficient for AF cell culture using our technique. Close agreement of the estimates of standard deviation of the preliminary and supplementary experiments (7.38 and 7.25) indicates that the assumptions required for our experimental design are correct and that our technical procedures are highly reproducible. A larger series of 76 AF specimens was used to correlate patient data with AF cell growth. We have shown that AF cell growth is independent of: (1) gestational age between 11 and 24 weeks; (2) maternal age between 14 and 42 years; (3) maternal ethnic group. A mean of 6 colonies/ml AF was obtained. Speculation: Development of highly efficient and reliable procedures for culturing AF cells depends upon statistically valid, quantitative assessment of all variables surrounding amniocentesis and subsequent cell culture. Using a randomized, multivariable block design, optimal culture procedures and conditions can be established and standardized which result in increased culture success rate and decreased time requirement for prenatal detection.