Self-assembled innervated vasculature-on-a-chip to study nociception

Abstract

Nociceptor sensory neurons play a key role in eliciting pain. An active crosstalk between nociceptor neurons and the vascular system at the molecular and cellular level is required to sense and respond to noxious stimuli. Besides nociception, interaction between nociceptor neurons and vasculature also contributes to neurogenesis and angiogenesis.In vitromodels of innervated vasculature can greatly help delineate these roles while facilitating disease modeling and drug screening. Herein, we report the development of a microfluidic-assisted tissue model of nociception in the presence of microvasculature. The self-assembled innervated microvasculature was engineered using endothelial cells and primary dorsal root ganglion (DRG) neurons. The sensory neurons and the endothelial cells displayed distinct morphologies in presence of each other. The neurons exhibited an elevated response to capsaicin in the presence of vasculature. Concomitantly, increased transient receptor potential cation channel subfamily V member 1 (TRPV1) receptor expression was observed in the DRG neurons in presence of vascularization. Finally, we demonstrated the applicability of this platform for modeling nociception associated with tissue acidosis. While not demonstrated here, this platform could also serve as a tool to study pain resulting from vascular disorders while also paving the way towards the development of innervated microphysiological models.

Keywords: innervation; organ-on-a-chip; tissue engineering; vascular tissue engineering.

Figure 1.

Development of innervated vasculature-on-a-chip. (A) Schematic depiction of the cell isolation and process of generating innervated vasculature tissue by co-culturing DRG neurons and endothelial cells (HUVECs). (B) Characterization of the innervated vascular network using immunostaining for endothelial (VE-cadherin) and neuronal markers (β-III tubulin) (C) 3D z-stack images of the tissue showing direct contact between the vasculature and neurites. White arrows show the co-localization of β-III tubulin and VE-cadherin Scale: 100 μm for (B) and 50 μm for (C).

Figure 2.

Reciprocal interaction between the endothelial cells and DRG neurons. (A) Representative images showing β-III tubulin staining of DRG neurons in the monoculture, co-culture without direct cell-cell contact (in adjacent microchannels), and co-cultures with direct cell contact. (B) Culture condition-dependent changes in number of neurons that sprouted neurites, number of neurites, and neurite length. N = 3–4 independent devices where each data point represents average of ≥ 5 ROIs from each independent device. (C) Phalloidin staining (red) of the innervated vascular network with β-III tubulin stained DRG neurons (green) (D) Characterization of average vessel diameter, branch density, and % coverage of the microvascular network under different culture conditions. N = 3–4 independent devices where each point represents an average of ≥ 3 ROIs from each independent device. *P < 0.05, **P < 0.01, ****P < 0.0001, n.s. (not significant) Scale: 100 μm. D: DRG monoculture; H: HUVEC monoculture; D&H: DRG and HUVEC non-contact co-culture; D+H: DRG and HUVEC direct contact co-culture.

Figure 3.

Presence of microvasculature sensitizes the neurons. (A) Representative images of Fura-2 ratiometric calcium imaging at varying capsaicin (Cap) concentration and KCl at 380 nm as well as corresponding ratiometric intensity (340 nm/380 nm). (B) Normalized average peak response curve for DRG neurons as a function of capsaicin concentration. N = 43 neurons for DRG+HUVEC co-culture condition and N=29 neurons for DRG monoculture condition (C) Normalized average peak response curve for responsive DRG neurons as a function of capsaicin concentration. N = 29 neurons for DRG+HUVEC co-culture condition and N=21 neurons for DRG monoculture condition. (D) Immunostaining for TRPV-1, CGRP and (E), (F) their quantification. N=49 neurons for DRG monoculture and N=80 for DRG+HUVEC co-culture. *P < 0.05, ***P < 0.001, Scale: 50 μm for (A) and 100 μm for (D).

Figure 4.

Modeling acidosis-induced nociception. (A) Representative ratiometric intensity images of Fura-2 at varying pH. White arrows point to cells whose ratiometric intensity changes compared to a higher pH. (B) Representative plot of average ratiometric response curve for DRG neurons as a function of varying pH. N=16 neurons. (C) Average peak ratiometric intensity of DRG neurons for varying pH. N= 11–16 neurons per pH condition. **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale: 50 μm

Figure 5.

Generation of innervated vasculature-on-a-chip using human DRG neurons (A) HUVECs and DRG neurons co-cultured within the device and imaged at Day 0 and Day 5, which shows the self-assembly of HUVECs into vasculature and neurites sprouting from the neurons. (B) Immunostaining of the innervated vasculature tissue for endothelial (VE-cadherin) and neuronal (β-III tubulin) markers. (B) β-III tubulin staining of the DRG neurons with and without the vasculature and quantification of (C) average neurite length and (D) number of neurites sprouted per cell. N=13 neurons for DRG monoculture and N=20 neurons for DRG+HUVEC co-culture (E) β-III tubulin and TRPV1 staining of the DRG neurons with and without the vasculature and (F) quantification of TRPV1 expression intensity. N=33 neurons for DRG monoculture and N=31 for DRG+HUVEC co-culture. ****P < 0.0001, n.s. (not significant) Scale: 100 μm