Practical Approaches to Improve the Sensitivity of the Volume-Based Cellular Enumeration of the BeWo Cell Line-Derived Spheroids as an In Vitro Embryo Model: MTT Versus Neutral Red Uptake Assays

Abstract

Objectives: Choriocarcinoma spheroids, as acceptable three-dimensional (3D) models of the embryo, vary in the number of incorporated cells based on the procedures of production. Since the secretion of emitted signals from spheroids can modulate endometrial confrontation responses, improving a high throughput standard assay is precisely demanding for normalizing the characteristics of spheroids based on cellular enumeration. Materials and Methods: Spheroids derived from the suspension culture of the BeWo cell line were dimensionally categorized into three groups with sizes of 50, 100, and 200 µm in diameters. The volume of each spheroid was determined according to 4/3πr3 formula and MTT and neutral red uptake (NRU) assays were applied as conventional and modified procedures. Furthermore, the categorized groups of spheroids were enzymatically dissociated and stained by the Trypan blue dye to precisely enumerate the number of incorporated cells based on volume alterations. Results: The absorbance of spheroids in modified MTT and NRU procedures with respect to the conventional ones resulted in higher correlation coefficients with volume alterations. Similarly, modified MTT and NRU procedures with respect to the conventional ones resulted in higher correlation coefficients between the cells numeration and volume alterations. Finally, the sensitivity of modified MTT assay was higher than modified NRU assay regarding accurately estimating the number of incorporated cells per spheroid. Conclusion: In general, the modification of MTT and NRU procedures can amplify the potential of both assays to enumerate the cell number of 3D aggregates. Exclusively, the modification of MTT assay brought it as an ideal high throughput assay with high sensitivity to screen the cell number of spheroids with a diameter of 200 µm and less.

Keywords

• cellular spheroids
• choriocarcinoma
• neutral red
• mtt formazan
• embryo implantation