Table of Contents
ISRN Obstetrics and Gynecology
Volume 2012 (2012), Article ID 152781, 6 pages
http://dx.doi.org/10.5402/2012/152781
Clinical Study

Preventing Microbial Contamination during Long-Term In Vitro Culture of Human Granulosa-Lutein Cells: An Ultrastructural Analysis

1Department of Obstetrics and Gynecology, Faculty of Medicine of Ribeirão Preto, University of São Paulo, 14049-900 Ribeirão Preto, SP, Brazil
2Department of Cell and Molecular Biology and Pathogenic Bioagents, Faculty of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil

Received 9 July 2012; Accepted 29 July 2012

Academic Editors: C. Iavazzo and J. G. Schenker

Copyright © 2012 C. O. Campos et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Purpose. To investigate whether the addition of antibiotic/antimycotic during human granulosa-lutein cells (GLCs) isolation and cell-plating procedures prevents microbial contamination after 144 h of culture and also evaluate the effects of contamination on GLCs ultrastructure and steroid secretion. Methods. GLCs obtained from five women submitted to assisted reproductive techniques (ARTs) were isolated with PBS supplemented with antibiotic/antimycotic or PBS nonsupplemented and cultured for 144 h. GLCs were evaluated by transmission electron microscopy (TEM), and estradiol (E2) and progesterone (P4) secretion was assayed by chemiluminescence. Results. Although no contaminating microorganisms were identified by light microscopy, TEM analyses revealed several bacterial colonies in culture dishes of GLCs isolated with only PBS. Bacterial contamination disrupted the adherence of the GLCs to the culture plate interfering with monolayer formation affecting the growth pattern of GLCs. Various cellular debris and bacteria were observed, and no organelles were found in the cytoplasm of infected cells. While bacterial contamination decreased estradiol media levels, it increased progesterone, as compared with noncontaminated group. Conclusion. Taken together, our data showed that the addition of a high dose of antibiotic/antimycotic during the isolation and cell-plating procedures prevents microbial contamination of long-term GLCs culture as its effects on cells growth and function in vitro.