Figure 1.
Key experimental steps in HIV pseudovirus production and challenges for automation.
(A) Summary of the key experimental steps of the manual procedure for the production of HIV-1 Env-pseudotyped viruses. (B) Definition of the main challenges to transfer the manual process to the automated system.
Figure 2.
Automated system for cell culture maintenance and HIV pseudovirus production.
(A) The complete system covered with a biosafety cabinet class II. (B) The robotic manipulator arm (RoMa) transports RoboFlasks from the incubator to the Flask Flipper at the worktable. (C) 2 RoboFlasks are fixed at the Flask Flipper and pierced by the needle of the Liquid Handling Arm (LiHa) for aspirating or dispensing liquids.
Figure 3.
Detailed workflow of the automated procedure.
(A) Cultivation of the 293T/17 cells and (B) the HIV-1 pseudovirus preparation with the adapted volumes of growth medium, PBS, Trypsin-EDTA, the incubation times and the cell numbers. (C) The performed manual steps.
Figure 4.
Accuracy of the measurements of cell density.
Illustrated are 10 measurements of a suspension of 293T/17 cells conducted by the Cedex Cell Counter and the Neubauer hemacytometer chamber. The values of Cedex counting are within 1.5-fold of the mean value of the measurements with the Neubauer hemacytometer chamber.
Figure 5.
Stable cell number of 293T/17 cells cultivated with the automated system.
Shown are 2 different maintenance runs (No. 2120, No. 2138) cultivated for 15 passages with the automated system. The cell numbers range for each splitting task between the ranges of 6×106 and 30×106 cells per harvested RoboFlask.
Table 1.
Viability of 293T/17 cells maintained over 6 passages on the automated system.
Table 2.
Quality control of the precision of large-scale HIV-1 pseudovirus stocks: Comparison of automated and manual produced viruses by neutralization titers with test reagents.
Table 3.
Quality control of the reproducibility: Comparison of the neutralization titers with test reagents of automated and manual produced HIV-1 pseudoviruses.