Perfusion of hLN-on-chip with lymphatic and blood
endothelial cells by coculture of human dermal lymphatic endothelial
cells or umbilical vein endothelial cells (HUVEC) will be achieved by
moving to a more complex chip design (Fig 6 below) where the LN stroma
can be perfused by a microengineered channel on either side.
The
channels are separated only by a slight constriction (Fig. 6B), thus
enabling cell-cell contact between the channels. To mimic in
vivo like vasculature, we will use the side channels to culture
lymphatic or vascular endothelium either side of the exposed surface
of the gel. We have shown that endothelial cells in such channels form
an in-vivo like monolayer with barrier function and active transport.
Further studies from other laboratories have identified the role of
hydrostatic pressure on the growth of lymphatic and vascular vessels.
Thus, we will optimize the hydrostatic pressure on the lymphocyte
containing gel to perfuse it with microvessels. With the help of
collaborators (Esak Lee, postdoctoral researcher in Chris Chen’s lab),
we can also induce lymph and vascular neoangiogenesis using cytokine
gradients within the lymph node stroma. The major caveat here is donor
mismatch which can be addressed by a) HLA typing or b) obtaining
matched patient tumor (as source of tumor cells and immune cells for
in Aim 3 and endothelial cells) and PBMC. If need be, we may limit our
experiments to contact independent interactions where the endothelium
is separated from the lymphocytes by a porous membrane (0.4-1uM pores
to prevent cellular traffic). However, we will start with endothelial
and PBMC samples from different donors as previously data have
successfully demonstrated HUVEC and PBMC culture from separate donors
(13).