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Non-Invasive Optical Sensor Based Approaches for Monitoring Virus Culture to Minimize BSL3 Laboratory Entry.

Ragupathy V, Setty MK, Kostov Y, Ge X, Uplekar S, Hewlett I, Rao G - Sensors (Basel) (2015)

Bottom Line: Routine PPE use involves significant recurring costs.Alternative non-invasive optical sensor based approaches to remotely monitor cell culture may provide a promising and cost effective approach to monitor infectious virus cultures resulting in lower disruption and costs.The replacement of culture media for cell and virus propagation and virus load monitoring was effectively performed using this fluorescent sensor and resulted in half the number of visits to the BSL3 lab (five versus ten).

View Article: PubMed Central - PubMed

Affiliation: LMV/DETTD/OBRR/CBER/FDA, Silver Spring, MD 20993, USA. viswanath.ragupathy@fda.hhs.gov.

ABSTRACT
High titers of infectious viruses for vaccine and diagnostic reference panel development are made by infecting susceptible mammalian cells. Laboratory procedures are strictly performed in a Bio-Safety Level-3 (BSL3) laboratory and each entry and exit involves the use of  disposable Personnel Protective Equipment (PPE) to observe cell culture conditions. Routine PPE use involves significant recurring costs. Alternative non-invasive optical sensor based approaches to remotely monitor cell culture may provide a promising and cost effective approach to monitor infectious virus cultures resulting in lower disruption and costs. We report here the monitoring of high titer cultures of Human Immunodeficiency Virus-1 (HIV-1) and Herpes Simplex Virus-2 (HSV-2) remotely with the use of optical oxygen sensors aseptically placed inside the cell culture vessel. The replacement of culture media for cell and virus propagation and virus load monitoring was effectively performed using this fluorescent sensor and resulted in half the number of visits to the BSL3 lab (five versus ten).

No MeSH data available.


Related in: MedlinePlus

HSV-2 infection monitoring of sensor patches. Each spike in the figure indicates a BSL-3 visit was made to replenish cells and collect an aliquot of culture supernatant for HSV-2 titer determination.
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sensors-15-14864-f002: HSV-2 infection monitoring of sensor patches. Each spike in the figure indicates a BSL-3 visit was made to replenish cells and collect an aliquot of culture supernatant for HSV-2 titer determination.

Mentions: The sensor was also evaluated for its correlation with HIV-1 viral load in terms of HIV-1 p24 levels. Before placing culture flasks over the electronic sensor, an aliquot of culture supernatant was collected that served as the day 0 sample. This experiment was carried out for 21 days and supernatants were collected at seven day intervals. HIV replication increased with time and a reduction in respiration of cells was observed. As the equilibrium was established by feeding cells at day 14 and 21, a rise in oxygen saturation was observed (Figure 1) with positive rate change. In contrast, in the uninfected flask, oxygen saturation decreased similar to the infected flask, likely due to masking of sensor patches with over grown cells. In order to keep the equilibrium constant, the culture medium was replenished at intervals when they achieved levels of oxygen below 50% or virus titers as high as 242 ngs/mL were observed at day 21. Finally, the sensor patch was also evaluated with a virus that infects its susceptible cells but does not cause its destruction upon replication. One such example is HSV-2. Productive infection is noted from a Taqman assay comparing day 4 (ct 28.6) and day 14 (ct 22.6) virus load (see Supplementary material). Similar to HIV, after a certain period of time, there was a decline in the oxygen concentration obtained with both infected and uninfected cells (Figure 2). Changes in the pH levels of the culture medium were also observed, indicating that cells were over grown and required a subculture. After the subculture, the cells again attained the maximum oxygen percentage.


Non-Invasive Optical Sensor Based Approaches for Monitoring Virus Culture to Minimize BSL3 Laboratory Entry.

Ragupathy V, Setty MK, Kostov Y, Ge X, Uplekar S, Hewlett I, Rao G - Sensors (Basel) (2015)

HSV-2 infection monitoring of sensor patches. Each spike in the figure indicates a BSL-3 visit was made to replenish cells and collect an aliquot of culture supernatant for HSV-2 titer determination.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4541811&req=5

sensors-15-14864-f002: HSV-2 infection monitoring of sensor patches. Each spike in the figure indicates a BSL-3 visit was made to replenish cells and collect an aliquot of culture supernatant for HSV-2 titer determination.
Mentions: The sensor was also evaluated for its correlation with HIV-1 viral load in terms of HIV-1 p24 levels. Before placing culture flasks over the electronic sensor, an aliquot of culture supernatant was collected that served as the day 0 sample. This experiment was carried out for 21 days and supernatants were collected at seven day intervals. HIV replication increased with time and a reduction in respiration of cells was observed. As the equilibrium was established by feeding cells at day 14 and 21, a rise in oxygen saturation was observed (Figure 1) with positive rate change. In contrast, in the uninfected flask, oxygen saturation decreased similar to the infected flask, likely due to masking of sensor patches with over grown cells. In order to keep the equilibrium constant, the culture medium was replenished at intervals when they achieved levels of oxygen below 50% or virus titers as high as 242 ngs/mL were observed at day 21. Finally, the sensor patch was also evaluated with a virus that infects its susceptible cells but does not cause its destruction upon replication. One such example is HSV-2. Productive infection is noted from a Taqman assay comparing day 4 (ct 28.6) and day 14 (ct 22.6) virus load (see Supplementary material). Similar to HIV, after a certain period of time, there was a decline in the oxygen concentration obtained with both infected and uninfected cells (Figure 2). Changes in the pH levels of the culture medium were also observed, indicating that cells were over grown and required a subculture. After the subculture, the cells again attained the maximum oxygen percentage.

Bottom Line: Routine PPE use involves significant recurring costs.Alternative non-invasive optical sensor based approaches to remotely monitor cell culture may provide a promising and cost effective approach to monitor infectious virus cultures resulting in lower disruption and costs.The replacement of culture media for cell and virus propagation and virus load monitoring was effectively performed using this fluorescent sensor and resulted in half the number of visits to the BSL3 lab (five versus ten).

View Article: PubMed Central - PubMed

Affiliation: LMV/DETTD/OBRR/CBER/FDA, Silver Spring, MD 20993, USA. viswanath.ragupathy@fda.hhs.gov.

ABSTRACT
High titers of infectious viruses for vaccine and diagnostic reference panel development are made by infecting susceptible mammalian cells. Laboratory procedures are strictly performed in a Bio-Safety Level-3 (BSL3) laboratory and each entry and exit involves the use of  disposable Personnel Protective Equipment (PPE) to observe cell culture conditions. Routine PPE use involves significant recurring costs. Alternative non-invasive optical sensor based approaches to remotely monitor cell culture may provide a promising and cost effective approach to monitor infectious virus cultures resulting in lower disruption and costs. We report here the monitoring of high titer cultures of Human Immunodeficiency Virus-1 (HIV-1) and Herpes Simplex Virus-2 (HSV-2) remotely with the use of optical oxygen sensors aseptically placed inside the cell culture vessel. The replacement of culture media for cell and virus propagation and virus load monitoring was effectively performed using this fluorescent sensor and resulted in half the number of visits to the BSL3 lab (five versus ten).

No MeSH data available.


Related in: MedlinePlus