Fig 6. Indirect 3-D co-culture setup that supports the growth of O. volvulus L4.

(A) FTSM and larvae are co-cultured in separate culture inserts with a schematic representation of hDF migration into the larvae compartment. (B) Image of the two inserts in a 6-well-culture plate containing the 3-D indirect co-culture setup. (C) SEM picture depicting cells that migrated inside the larvae containing insert and grew over the viable O. volvulus L4. Inset shows the interface between L4, cells, and ECM. Scale bar = 20 μm. (D) Growth of 15-days old L4 co-cultured in the indirect 3-D setup using two experimental co-culture media, 3D-3 and 3D-4, versus 2D-ctrl (over a HUVEC monolayer) over 77 days of culture (observations reported are those for day 15, 36, and 92 of worm age). Each experimental condition was set up with 3 technical replicates containing ~10 L4s each. The % motile L4s in each group and time point are indicated below the graph. The significance of differences in growth between days in culture was analyzed using the Mann-Whitney U test (P < 0.0001: ****), and between the groups on day 77 of culture using ANNOVA with Dunnet’s analysis (P ≤ 0.001: ***; P < 0.0001: ****)