Online monitoring of the baculovirus expression vector system in bioreactors

Background

The increasing threat of pandemics due to the appearance of new viral diseases and the increased spread of existing viruses globally urges the development of a flexible vaccine production platform. Virus like particles (VLPs) form a promising new vaccine concept and can be produced using the baculovirus expression vector system and insect cells. Online determination of the viable cell density and state of infection can help in determining the optimal parameters for the production process.
The iLine F microscope system continuously pumps suspension cells in culture through a measuring chamber in a closed-loop system and makes holographic images of the cells resulting in up to 70 parameters per individual cell. Thus, it can accurately determine cell concentration and cell shape. The next step is to study whether it can also measure the infection state of the cell.

Objective

Evaluate online 3D holographic microscopy (iLine F) to determine the fraction of infected cells.

Results

Figure 2: The total and viable cell density determined by the iLine F (red lines) and offline counts (square markers).

Figure 3: The viability determined by the iLine F (solid red) and trypan blue staining (square markers).

Figure 4: mCherry fluorescence measured with flow cytometer. Cells with values higher than the gate (dotted black line) were determined to be infected. Sample timepoints are given in hours post infection (HPI).

Figure 5: Fraction of infected cells determined by the iLine F (solid red) and the flow cytometer (square markers). AcBAC-PH-mCherry (MOI=0.1) was added to the reactor at 73 hours post inoculation (dotted black line).

Figure 1. A: A single use bioreactor setup with the iLine F monitoring system. B: The cell segmentation and live dead determination performed by the microscope (green markers: live, red markers: dead). C: The 3D image created by the microscope.

Methods

• ExpiSf cells (Thermo Fisher) were cultured in ExpiSf chemically defined medium (Gibco)
• AcMNPVs: AcBAC-PH-HisGFP, AcBAC-PH-mCherry
• Applikon MiniBio reactors (400mL working volume, DO=30%, T=27⁰C)
• iLine F microscope system (OVIZIO)
• TC20 automated cell counter (Bio-Rad)
• Cells were infected at 3-4×10^6 cells/mL with a multiplicity of infection of 0.1 (TCID/cell)

Figure 6: The 3D image made by the iLine F of a non-infected and infected Sf9 cell.

The iLine F showed a cell density and viability trend similar to the offline measurements. Figure 5 shows that the iLine F was able to detect baculoviral infection earlier than the offline flow cytometer method. Since mCherry was expressed behind the very late polyhedrin promoter (active at ~18 hours post infection) the flow cytometer method shown in Figure 4 and 5 only detected cells that were in the late stage of infection. Online holographic microscopy can capture changes directly as they occur on the cellular level, eliminating the need of offline sample preparation and reporter proteins such as mCherry. Detection is based on a combination of parameters generated by the microscope. A training of the algorithm was necessary (data not shown). With increasing amounts of data the algorithm can be further optimized and also used to re-analyze old data sets.

Conclusion

  • The iLine F microscope system is able to accurately estimate the fraction of baculoviral infected cells.

  • Cell infection can be detected in real time and earlier than offline methods.

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Maarten Klaverdijk¹, Jort Altenburg¹, Dirk Martens¹, Gorben Pijlman², Jan van Hauwermeiren³, Jérémie Barbau³, Laurent Desmecht³, Damien Cabosart³

1. Bioprocess Engineering, Wageningen University and Research, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands,
2. Laboratory of Virology, Wageningen University and Research, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands,
3. Ovizio Imaging Systems, Rue du Bourdon 100/2 1180 Brussels, Belgium


Examination of vesicular stomatitis virus-induced morphology changes in individual Vero cells by qMod microscopy

Viral vectors are increasingly gaining importance in vaccine development, gene therapy and as oncolytic vectors. Vesicular stomatitis virus (VSV), an enveloped virus carrying a negative-sense RNA genome, has proven to be an excellent vaccine vector candidate against infectious diseases and specific cancers.

A variety of assays are available to determine yield of physical particles as well as infectious particles. However, several of the most well-characterized methods, such as plaque assays and tissue culture (TC) infectious dose 50 (TCID50) assays, are associated with a wide variance and are also time- and labor-intensive. Therefore, rapid and reliable means of assaying virus products are welcome advances, especially if the assay easily adapts to multiple product lines.

In this report, we describe a novel viral infection analysis method using a live VSV-based Lassa virus (LASV) vaccine candidate. The recombinant VSV has been genetically altered to express the LASV Josiah glycoprotein (VSVΔG/LASVGP), and infection in Vero cells was examined by microscopy using the Ovizio qMod camera and OsOne software. We assessed characteristics of infected Vero cells over the course of infection, obtained feature measurements of individual cells and examined very early biophysical distinctions of infected cells.

Process Development, Ology Bioservices, Alachua, FL 32615, USA
Isabel Scholz*, Christopher Montoya & Eric Vela

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Digital holographic microscopy: a novel tool to study the morphology, physiology and ecology of diatoms.

Eva-Maria Zetsche, Ahmed El Mallahi & Filip J. R. Meysman. Diatom Research, 31:1, 1-16, 2016.

A recent publication by Zetsche et al. (2016) highlights the difficulties in imaging aquatic organisms such as diatoms: “Diatom cells are for the large part transparent, and since transparent substances or objects, by definition, do not absorb light in appreciable quantities when suspended in water, these entities are hard to discriminate and detect by microscopy techniques that rely on intensity information alone”.

“Piper (2011) suggested that interference-based contrast microscopy reveals the shape and structure of cells more clearly, as it improves the plasticity and contour sharpness.” Digital holographic microscopy (DHM) is in fact an interference-based approach and Dr. Zetsche and her co-authors are able to show that with DHM “the structural organization of diatoms is more clearly determined, in terms of cellular components, shapes and features.”

Ovizio’s qMod, a differential digital holographic microscopy camera for classical microscopes, is one of the instruments which offers a tool for the improved discrimination of living and dead diatoms (success rate >95%). Possible future applications can be the live-dead discrimination of microscopic aquatic organisms, as well as improved species identification. “Certain species of diatoms are frequently used to assess the water quality of rivers and lakes as well as coastal areas (Anton-Garrido et al. 2013, Kelly et al. 2009, Sabater et al. 2007). DHM may facilitate the live-dead differentiation of cells and thus improve these monitoring procedures” (Zetsche et al. 2016).

Figure: (a) Hologram of a cleaned frustule of  Stauroneis  sp. (University of Gent, Belgium) as obtained with an Ovizio digital holographic microscope. This hologram contains both light-intensity information (b) as well as phase information (c) representing the optical path length (OPL) of the object. (d) The OPL of an object is more clearly visualized with false coloring of the phase information. (Taken from Zetsche et al. 2016)

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