Innovative Approach to Fast Electron Microscopy Using the Example of a Culture of Virus-Infected Cells: An Application to SARS-CoV-2

Abstract

Despite the development of new diagnostic methods, co-culture, based on sample inoculation of cell monolayers coupled with electron microscopy (EM) observation, remains the gold standard in virology. Indeed, co-culture allows for the study of cell morphology (infected and not infected), the ultrastructure of the inoculated virus, and the different steps of the virus infectious cycle. Most EM methods for studying virus cycles are applied after infected cells are produced in large quantities and detached to obtain a pellet. Here, cell culture was performed in sterilized, collagen-coated single-break strip wells. After one day in culture, cells were infected with SARS-CoV-2. Wells of interest were fixed at different time points, from 2 to 36 h post-infection. Microwave-assisted resin embedding was accomplished directly in the wells in 4 h. Finally, ultra-thin sections were cut directly through the infected-cell monolayers. Our methodology requires, in total, less than four days for preparing and observing cells. Furthermore, by observing undetached infected cell monolayers, we were able to observe new ultrastructural findings, such as cell–cell interactions and baso-apical cellular organization related to the virus infectious cycle. Our innovative methodology thus not only saves time for preparation but also adds precision and new knowledge about viral infection, as shown here for SARS-CoV-2.

Limited permissibility of ENL-R and Mv-1-Lu mink cell lines to SARS-CoV-2

“…The morphological changes promoted by the viral infection often trigger the cytopathic effect ( Albrecht et al, 1996 ), which, although it was not visible when observing the monolayer of cells in ordinary light microscopy, in electron microscopy, the infected cells were often lysed after infection, for both Mv-1-lu ( Figures 3C – F ) and ENL-R ( Figure 4A ) cells. The SARS-CoV-2 particles were identified based on previous electron microscopy morphological studies from our laboratory and other groups ( Brahim et al, 2020 ; Colson et al, 2020 ; Laue et al, 2021 ; Le Bideau et al, 2021 ).…”

“…The infected wells were fixed on days 0, 1, 2, and 7 d.p.i with the addition of 20 μL of 25% glutaraldehyde. The wells thus fixed were prepared with a microwave-assisted resin coating directly in the wells, and ultra-thin sections were cut straight through the monolayers of the infected cells and then observed with SU5000 SEM (Hitachi High-Technologies, HHT, Japan; Le Bideau et al, 2021 ). This electron microscopic observation was performed on SARS-CoV-2 strains of groups B.1 and B.1.160.…”

“…This electron microscopic observation was performed on SARS-CoV-2 strains of groups B.1 and B.1.160. In addition, scanning electron microscopy of ultra-thin sections of Mv-1-Lu and ENL-R cell monolayer was performed according to the novel approach described by Le Bideau et al (2021) .…”

Control of CDH1/E-Cadherin Gene Expression and Release of a Soluble Form of E-Cadherin in SARS-CoV-2 Infected Caco-2 Intestinal Cells: Physiopathological Consequences for the Intestinal Forms of COVID-19

“…Virus-free cells and SARS-CoV-2 infected Caco-2 cells at 48 hours post-infection were prepared as previously described ( Le Bideau et al., 2021 ). Briefly, cells were fixed with glutaraldehyde (2.5%) in a 0.1 M sodium cacodylate buffer.…”

Microscopic observations of SARS‐CoV‐2 like particles in different oral samples

“…Saliva samples were deposited in Greiner Bio‐One 96‐well single‐break strip microplates (Greiner Bio‐One) coated with poly‐L lysine to retain the material. Resin embedding of the microplates was then performed as previously described [ 14 ]. Resin embedding was microwave‐assisted with PELCO BiowavePro+ (Ted Pella).…”