2.2 Inoculation of Cells With SARS-CoV-2 and Adventitious Agent
Testing
Cells (CHO, Vero, HEK-293, HT-1080 and MRC-5) in 6-well plates were
incubated with SARS-CoV-2 for 14 days. Inoculations with virus were done
at multiplicity of infection (MOI)=0.01 and MOI=1, that is, approx.
103 and 105 infectious virus
particles per well, respectively, for 1 h. Then the respective medium
was added to a total volume of 6 mL/well
(CHO medium: Ham’s F12 medium
supplemented with 10% FCS [PAN-Biotech P40-2009], L-glutamine
[2mM], nonessential amino acids [1x], sodium pyruvate [1mM]
and Gentamycin sulfate [100 mg/ml]); Vero medium: TC-Vero medium
supplemented with 5% FCS, L-glutamine [2mM], nonessential amino
acids [1x], sodium pyruvate [1mM], Gentamycin sulfate [100
mg/ml] and sodium bicarbonate [7.5%]; HEK 293 medium: EAGLE-MEM
(+Earles BSS) supplemented with 5% FCS and Gentamycin sulfate [100
mg/ml]; HT-1080 medium: McCoy 5A supplemented with 2% FCS ,
L-glutamine [2mM], sodium pyruvate [1mM] and Gentamycin sulfate
[100 mg/ml]; MRC-5 medium: EAGLE-MEM (+Earles BSS) supplemented with
10% FCS, L-glutamine [2mM], nonessential amino acids [1x],
sodium pyruvate [1mM],Gentamycin sulfate [100 mg/ml] and sodium
bicarbonate [7.5%]). 6-well plates were incubated for 14 days at
36°C and 4.5% CO2. The presence of cytopathological
effects (CPE) was assessed on days 2, 3, 6, 7 and 14 post-infection.
The presence of infectious SARS-CoV-2 was tested by titration at various
stages throughout the incubation period, on Vero cells using a
TCID50 assay (see below). Samples for titration were
taken from the input inoculum and days 2 (Vero cells MOI=1 only), 3, 6,
7 and 14 post-infection.
For each cell line, inoculations with SARS-CoV-2 at the two MOIs was
done in duplicate.
In addition to SARS-CoV-2 readout by CPE, after 14 days of inoculation,
hemadsorption (HAD) and hemagglutination (HA) assays were done, as
routinely performed during AAT.
For HAD, wells with MRC-5, Vero and CHO cells were covered with
erythrocyte suspensions of two different species (human 0.5% [v/v]
and guinea pig 1% [v/v]). Separate plates were incubated at +2–8°C
and 36°C for 30 min, the cell culture supernatant removed, and the cells
washed twice with PBS before microscopic inspection for HAD.
For HA, supernatants of MRC-5, Vero, CHO, HEK-293 and HT-1080 cells were
diluted with 0.9% [w/v] NaCl solution in twofold steps. Erythrocyte
suspensions (human, 0.5% [v/v] and guinea pig 1.0% [v/v]) were
added and separate V-shaped plates incubated for 35 min at +2–8°C and
36°C, before hemagglutination was inspected visually. The described AAT
is essentially the routine procedure used for the testing of recombinant
protein bulk harvests.