Liquid handling system

Viral neutralization by trial participant serum samples were measured using single-round HIV-1 Env pseudovirus infection of TZM-bl target cells. Env-pseudotyped viruses were generated by transfection of 293T/17 cells with optimized ratios of envelope-expressing plasmid and backbone vector (pSG3DEnv). A panel of three viruses was tested for neutralizing activity spanning a range of 80% inhibitory concentrations (IC₈₀) for bnMAb VRC01. Negative and positive controls were included in all assays. The tested viruses were Q23.17 (subtype A), PVO.04 (subtype B), and MW965.26 (subtype C). Serum neutralization assays were performed in 384-well plates using a Beckman Biomek liquid handling system, as previously described [33 (link)]. Experimental results were displayed as the serum dilution that produced 80% neutralization (ID₈₀) against the viruses tested. Predicted ID₈₀ values were calculated based on the concentration of VRC01LS present in the sera and the established inhibitory concentration (IC₈₀) of the antibody against each virus.

As a control for our AMOs, we generated cortical hiPSC organoids from the same hiPSC line used to derive smNPCs for AMO line 2 (Reinhardt et al., 2013b (link)). After manual 2D culture of hiPCs, all steps were fully automated using our liquid handling system (Beckman Coulter) with attached incubator (Thermo Fisher). Generally, we followed the protocol previously published by Paşca et al., 2015 (link). (and also described in more detail by Sloan et al., 2018 (link)), with adaptations for our automation pipeline (see Figure 7—figure supplement 1). Starting with 90–100% confluent cultures, we detached hiPSCs with accutase and seeded 10,000 cells per well in ultra-low attachment U-bottom plates (Corning). Cortical organoid medium consisted of DMEM F-12, 20% Knock-out Serum replacement (GIBCO), 1% penicillin/streptomycin/glutamine, 1% Non-essential amino acids (Sigma-Aldrich), and 0.2% 2-Mercaptoethanol (Thermo Fisher). For the first 6 days, we supplemented the cortical organoid medium with 5 μM dorsomorphin (Enzo Life Sciences) and 10 μM SB-431542 (Biomol). During seeding only, we also added 10 μM ROCK inhibitor Y-27632. Aggregates were fed every 3 days using an automated liquid handling system. From day 6 to 24, culture medium supplements were exchanged to EGF and FGF2 (both 20 ng/ml, PeproTech) and afterwards BDNF and NT3 (metabion) (both 20 ng/ml).