title: The use of image analysis to evaluate the development of uterine and oviduct epithelial cells during in vitro culture. A potential quality assurance procedure for in vitro laboratories.
abstract: The objective of this study was to establish selection criteria for morphologic assessment of cell quality using a computer image-analysis system. Uterine and oviduct epithelial cells were isolated from the reproductive tracts of cyclic cows using a trypsin solution. Harvested cells were cultured in Tissue Culture Medium-199 with 10% fetal bovine serum and maintained at 37 degrees C with 5% carbon dioxide in air. Subcultures of different confluent monolayers were established using a weak trypsin solution. Morphologic assessment of cell integrity and viability were made visually, with electron microscopy and image analysis. After morphologic assessment, cells were assigned quality scores. Evaluations were conducted on cells during primary culture and following the first and third subpassages. Image-analysis evaluation was conducted on cells using a silicon-intensified target camera and computer-based software. Gray-level density was determined on cells from each flask. There was no difference (P greater than .05) between sample replicates, indicating repeatable measurements were obtained with the image-analysis system. In addition, there was no significant difference in parameters measured between uterine and oviduct cells; therefore, data from both cell types were combined for further statistical comparisons. Gray-level density values for combined uterine and oviduct cells during primary culture and following the first and third subpassages were as follows: 139.7, 154.5, and 173.3, respectively. There was an increase (P less than .05) in gray-level density values with each subpassage of the cell populations. Furthermore, gray level was influenced (P less than .01) by cell quality for combined uterine and oviduct cell populations. These results indicate that epithelial cell quality during in vitro culture may be effectively determined using image analysis. This approach should not be overlooked in establishing quality control measures for uterine and oviduct cells before mammalian embryos are cocultured in vitro.
authors.affiliation: Department of Dairy Science, Louisiana State University, Baton Rouge 70803.
authors.forename: J K
authors.initials: JK
authors.lastname: Thibodeaux
authors.forename: J D
authors.initials: JD
authors.lastname: Roussel
authors.forename: K L
authors.initials: KL
authors.lastname: White
authors.forename: J R
authors.initials: JR
authors.lastname: Broussard
authors.forename: R A
authors.initials: RA
authors.lastname: Godke
mesh_terms: D000818:Animals; D002417:Cattle; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D004847:Epithelial Cells; D004848:Epithelium / Q000502:physiology / Q000648:ultrastructure; D005260:Female; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D008855:Microscopy, Electron, Scanning; D010057:Oviducts / Q000166:cytology* / Q000502:physiology / Q000648:ultrastructure; D014599:Uterus / Q000166:cytology* / Q000502:physiology / Q000648:ultrastructure