Detection of baculovirus-infected insect cells by flow cytometric side-scatter analyses

Nordström, Tommy; Willamo, Petra; Arvela, Mikaela; Stenroos, Karolina; Lindqvist, Christer

doi:10.1002/(sici)1097-0320(19991101)37:3<238::aid-cyto11>3.0.co;2-9

Cited by 10 publications

(9 citation statements)

References 13 publications

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“…Correlations between either the forward scatter (FSC) or side scatter (SSC) measurements with human adenovirus titers in HEK293 have been established (Sandhu and AlRubeai 2008). Other studies showed that the SSC parameter can be used to monitor baculovirus infection processes (Deparis et al 2003;Nordstrom et al 1999). In this work, a positive correlation was obtained between either the fluorescence intensity or the SSC measurement and maximum productivity ( Fig.…”

Section: Discussionsupporting

confidence: 68%

Production of canine adenovirus type 2 in serum-free suspension cultures of MDCK cells

Fernandes

Laske

Sousa

et al. 2015

Appl Microbiol Biotechnol

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The potential of adherent Madin Darby Canine Kidney (MDCK) cells for the production of influenza viruses and canine adenovirus type 2 (CAV-2) for vaccines or gene therapy approaches has been shown. Recently, a new MDCK cell line (MDCK.SUS2) that was able to grow in suspension in a fully defined system was established. In this work, we investigated whether the new MDCK.SUS2 suspension cell line is suitable for the amplification of CAV-2 under serum-free culture conditions. Cell growth performance and CAV-2 production were evaluated in three serum-free media: AEM, SMIF8, and EXCELL MDCK. CAV-2 production in shake flasks was maximal when AEM medium was used, resulting in an amplification ratio of infectious particles (IP) of 142 IP out/IP in and volumetric and cell-specific productivities of 2.1 × 10(8) IP/mL and 482 IP/cell, respectively. CAV-2 production was further improved when cells were cultivated in a 0.5-L stirred tank bioreactor. To monitor infection and virus production, cells were analyzed by flow cytometry. A correlation between the side scatter measurement and CAV-2 productivity was found, which represents a key feature to determine the best harvesting time during process development of gene therapy vectors that do not express reporter genes. This work demonstrates that MDCK.SUS2 is a suitable cell substrate for CAV-2 production, constituting a step forward in developing a production process transferable to industrial scales. This could allow for the production of high CAV-2 titers either for vaccination or for gene therapy purposes.

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Section: Discussionsupporting

confidence: 68%

Production of canine adenovirus type 2 in serum-free suspension cultures of MDCK cells

Fernandes

Laske

Sousa

et al. 2015

Appl Microbiol Biotechnol

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“…However, these researchers used methods other than FC to measure cell size and to my knowledge flow cytometric measurement of cell size in relation to productivity in virus cell systems has not been reported. Nordström et al (13) reported difficulty using flow cytometric measurement of cell size to monitor baculovirus infection; however, we have demonstrated a good and successful correlation between r‐protein production in a BEVS and flow cytometric FS (34). Monitoring of virus productivity with FC (FS parameter) will prove to be of immense practicability when no marker or reporter proteins are available, and as indicated before, such proteins are not desirable in vectors destined for human therapeutics.…”

Section: Resultsmentioning

confidence: 61%

“…The nucleus and cytoplasm will contain the assembled virions and so should demonstrate change in granularity that could also be monitored by the flow cytometric side scatter (SS). In the literature there are examples where side scatter was used to monitor baculovirus propagation in insect cells (11)(12)(13)(14). To our knowledge there is no reported use of granularity of Ad packaging cells as a parameter to monitor Ad infection.…”

Section: Introductionmentioning

confidence: 99%

Monitoring of the Adenovirus Production Process by Flow Cytometry

Sandhu

Al‐Rubeai

2008

Biotechnol. Prog.

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Adenovirus (Ad) has become the vector of choice for gene therapy clinical protocols worldwide; it is the only viral vector to date that has been licensed for use in a gene therapy treatment. There is, however, a need to develop a simple, reliable at-line method to monitor the production of virus and recombinant proteins (r-proteins) that have no intrinsic reporter properties. Here we utilize flow cytometry to measure cell size, granularity, and DNA content in a single-step analysis and to correlate these parameters to the production of a type-5 Ad (Ad5) expressing the recombinant green fluorescent protein (GFP). Clear correlations between these parameters and productivity are made, with forward scatter and DNA content showing the highest correlation coefficients, 0.9 and 0.83 for virus production and r-protein production, respectively. Measuring these parameters requires little or no processing of the cells from culture to analysis. These parameters have been used successfully to monitor, at-line, the amount of Ad and r-protein product in a 293-Ad system.

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“…The degree of side scatter on a flow cytometry plot is related to the cytoplasmic complexity of the cell (i.e., shape of the nucleus, the amount and type of cytoplasmic granules or the membrane roughness). In essence, side scatter is a measure of the relative granularity of the cell and thus, can be used as an indication of its health 16–19. An extremely attractive benefit of flow cytometry analysis is the ability to use selected cell surface markers to discriminate host (recipient) cells that may contaminate the encapsulated cells upon their retrieval, thus increasing the accuracy of this technique.…”

Section: Introductionmentioning

confidence: 99%

Characterization of viability and proliferation of alginate‐poly‐L‐lysine–alginate encapsulated myoblasts using flow cytometry

et al. 2010

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Genetically modified cells encapsulated in alginate-poly-L-lysine-alginate (APA) are being developed to deliver therapeutic products to treat a variety of diseases. The characterization of the encapsulated cells thus becomes paramount. This study reports a novel method to assess the viability, granularity and proliferation of encapsulated cells based on flow cytometry. The in vitro viability of encapsulated G8 murine myoblasts secreting canine FVIII (cFVIII) measured by flow cytometry was comparable to the traditional trypan blue exclusion method and both correlated with cFVIII secretion levels. In contrast, after implantation into mice, only viability measured by flow cytometry correlated with cFVIII secretion. Further, flow cytometry analysis of encapsulated cells maintained in vitro and in vivo revealed a greater fraction of granular cells compared to free cells, suggesting that encapsulation influences the morphology (cytoplasmic composition) of cells within APA microcapsules. Interestingly, the proliferation study showed that encapsulated cells proliferate faster, on average, and were more heterogeneous in vivo compared to in vitro culture conditions, suggesting that encapsulated cell proliferation is complex and environment-dependent. In conclusion, we show that flow cytometry analysis allows for a more consistent and comprehensive examination of encapsulated cells to aid in the development of cell therapy protocols.

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Detection of baculovirus-infected insect cells by flow cytometric side-scatter analyses

Cited by 10 publications

References 13 publications

Production of canine adenovirus type 2 in serum-free suspension cultures of MDCK cells

Production of canine adenovirus type 2 in serum-free suspension cultures of MDCK cells

Monitoring of the Adenovirus Production Process by Flow Cytometry

Characterization of viability and proliferation of alginate‐poly‐L‐lysine–alginate encapsulated myoblasts using flow cytometry

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