Non-invasive Visualization and Characterization of Bile Canaliculus Formation Using Refractive Index Tomography.

The vital role of bile canaliculus (BC) in liver function is closely related to its morphology. Electron microscopy has contributed to understanding BC morphology; however, its invasiveness limits its use in living specimens. Here, we report non-invasive characterization of BC formation using refractive index (RI) tomography. First, we investigated and characterized the RI distribution of BCs in two-dimensional (2D) cultured HepG2 cells. BCs were identified based on their distinct morphology and functionality, as confirmed using a fluorescence-labeled bile acid analog. The RI distribution of BCs exhibited three common features: (1) luminal spaces with a low RI between adjacent hepatocytes; (2) luminal spaces surrounded by a membranous structure with a high RI; and (3) multiple microvillus structures with a high RI within the lumen. Second, we demonstrated the characterization of BC structures in a three-dimensional (3D) culture model, which is more relevant to the in vivo environment but more difficult to evaluate than 2D cultures. Various BC structures were identified inside HepG2 spheroids with the three features of RI distribution. Third, we conducted comparative analyses and found that the BC lumina of spheroids had higher circularity and lower RI standard deviation than 2D cultures. We also addressed comparison of BC and intracellular lumen-like structures within a HepG2 spheroid, and found that the BC lumina had higher RI and longer perimeter than intracellular lumen-like structures. Our demonstration of the non-destructive, label-free visualization and quantitative characterization of living BC structures will be a basis for various hepatological and pharmaceutical applications.

A quantitative assessment of the morphological characteristics of BeWo cells as an in vitro model of human trophoblast cells / Una evaluación cuantitativa de las características morfológicas de las células BeWo como un modelo in vitro de las células de trofoblasto humano

In order to study the detailed morphology of trophoblast cells during human implantation, BeWo cells were cultured as spheroids in suspension culture. These cultures were then processed for light and electron microscopical examination. The present study showed that the BeWo spheroids consist of two cell types which are cytotrophoblast-like and syncytiotrophoblast-like. The cells with larger nuclear diameter made up only about 1 percent of the cell population and appear to be those of syncytiotrophoblast. Therefore the predominant cell type of the BeWo spheroids appeared to be relatively undifferentiated and cytotrophoblast-like. About 10 percent of the BeWo cells in the present study were mitotic, indicating a highly proliferative population. Total cell number increased about 12 times during the culture period from 107 +/- 9 on day 1 to 1211 +/- 145 on day 7 whereas the volume per cell increased about 2 times, from 1300 um3 on day 1 to 2400 um3 on day 7. Therefore overall growth of BeWo spheroids is due to both hyperplasia and hypertrophy. However, it appears that cell proliferation outstrips volumetric growth. These quantitative data show that BeWo cells grow mainly by hyperplasia and provide baseline values for further studies. In addition, the results show that BeWo cell morphology has marked similarities to that reported for human trophoblast, making it a useful model for subsequent in vitro studies.

En un cultivo de suspensión se estudió la morfología de las células durante la implantación del trofoblasto humano, células BeWo. Estos cultivos fueron procesados y examinados a través de microscopía de luz y electrónica. El estudio mostró que los esferoides BeWo constan de dos tipos de células, citotrofoblasto y sincitiotrofoblasto. Las células con mayor diámetro nuclear parecen ser los sincitiotrofoblasto que representaban sólo el 1 por ciento de la población celular. Por tanto, el tipo celular predominante de los esferoides BeWo parecían ser relativamente indiferenciados como citotrofoblasto. Alrededor del 10 por ciento de las células BeWo fueron mitóticas, lo que indica una población altamente proliferativa. El número de células totales aumentó alrededor de 12 veces durante el período de cultivo de 107 +/- 9 días en el día 1 a 1211 +/- 145 en el día 7, mientras que el volumen de la célula creció alrededor de 2 veces, desde 1300 mm3 el día 1 hasta 2400 mm3 el día 7. Por lo tanto, el crecimiento global de esferoides BeWo se debe tanto a la hiperplasia como a la hipertrofia. Sin embargo, parece que la proliferación celular supera al crecimiento volumétrico. Estos datos cuantitativos muestran que las células BeWo crecen principalmente por hiperplasia y proporcionan valores de referencia para estudios posteriores. Además, los resultados muestran que la morfología celular BeWo ha marcado similitudes con los reportado para el trofoblasto humano, por lo que es un modelo útil para posteriores estudios in vitro.