(69a) Engineering a Physiologically Relevant Model of the Cardiac Autonomic Nervous System

Authors: 
Soucy, J., Northeastern University
Torregrosa, T., Northeastern University
Hosic, S., Northeastern University
Annabi, N., Northeastern University
Koppes, A., Northeastern University
Koppes, R., Northeastern University

Engineering
a Physiologically Relevant Model of the Cardiac Autonomic Nervous System

Jonathan R Soucy1,
Tess Torregrosa1, Sanjin Hosic1, Nasim Annabi1,2,
Abigail N Koppes1,3, Ryan A Koppes1

1Chemical
Engineering, Northeastern University, Boston, MA

2Harvard-MIT
Division of Health Sciences and Technology, Massachusetts Institute of
Technology, Cambridge, MA

3Biology,
Northeastern University, Boston, MA

Introduction:

The
autonomic nervous system (ANS) functions to maintain homeostasis in the heart
via the complementary functions of the sympathetic nervous system (SNS) and the
parasympathetic nervous system (PSNS) [1].
However, ANS dysfunction can lead to increased cardiac arrhythmias and even
sudden cardiac failure due to an inability to effectively modulate heart hate following
overexertion or excessive stress [2-4].
Typically, dysautonomia arises to meet the increased cardiac demands of a
damaged myocardium by promoting SNS hyperinnervation/activation and PSNS
withdrawal/deactivation [1,
5, 6].
This understanding of the cardiac ANS pathophysiology has led to development of
beta-blocker therapies to inhibit SNS activity systemically [7-9],
and an investigation into vagal nerve stimulation to increase PSNS function [10-13].
Yet, there is no clear understanding of how to mediate an ANS imbalance, nor
are the underlying cellular mechanicals for cardiac innervation well understood.
Traditionally,
animal models have been used to investigate cardiac ANS dysfunction, but due to
their inherent complexities and variability, in vitro alternatives must
be developed [14].

Microfluidic
devices are an attractive platform to develop innervated muscle
organ systems in vitro, but only recently have
been applied to cardiac system [14-16].
In these previous works, the authors chose to develop models focused primarily
on compartmentalization and the hierarchical structure of the neuro-cardiac
axis, rather than a more physiologically relevant 3D cell culture, which may be
necessary to mimic cardiac innervation in vivo [17].
Therefore, to better recapitulate the in vivo environment, we aim to engineer
a microphysiological system to culture cardiac cells and ANS neuron populations
in a biomimetic scaffold.

Methods:

Custom microfluidic chips were fabricated using
a novel “laser cut and assembly” method via a commercial laser engraver system
to cut and shape acrylic sheets, double sided adhesive, and a polycarbonate
track etched membrane to support the 3D culture of cardiac cells and both SNS
and PSPS neurons (Figure 1). The devices utilize a phase guide to compartmentalize
cell-laden 3D hydrogels in the basal channel, and polycarbonate membrane to
enable medium diffusion from the apical channel and mimic circulation. Polycarbonate
provides benefits in minimizing analyte adsorption compared to traditional PDMS
platforms [18].
Primary
neonatal rodent cardiac cells from the heart, cholinergic neurons from the intracardiac
ganglia, and adrenergic neurons from the superior
cervical ganglia
will be isolated to develop our model of the cardiac ANS [14].
Cells will be encapsulated within a photocrosslinkable
gelatin based hydrogel, gelatin methacrylate (GelMA)
in situ using our
custom microfluidic chips. Specifically, GelMA will be crosslinked in the
presence of lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP, BioBots)
using an in-house LED photocrosslinking system (405nm). Cardiac output within
the innervated systems will be assessed with a custom MATLAB code to calculate
beating on a cell-by-cell basis using video microscopy, and results compared to
non-innervated cardiac cultures.

Results and Discussion:

Towards
our goal of recapitulating the ANS innervation into cardiac tissue,
using a commercially available chip (DAX1, AimBiotech), we have successfully encapsulated
cardiac cells in a biomimetic gelatin scaffold in situ and observed SNS
innervation (Figure 2). However, this commercially available
microfluidic system does not support the 3D encapsulation and compartmentation
of the neural components of the cardiac ANS. Therefore, we have fabricated a
custom laser cut microfluidic chip to support 3D compartmentalized cell culture
of cardiac cells and both ANS neuron populations. Notably, this device design permits
neurons to be encapsulated the day prior to the addition of cardiac cells,
which allows for the necessary handling time of each cell population. Additionally,
our custom chip contained a tight and well-defined hydrogel boundary between
compartments so that innervation will be unobstructed and easier to quantify
(not shown).

We have demonstrated
an ability to measure cardiac output (beat rate and beating synchrony) of
encapsulated cardiac cells cultured in microfluidics chips using video
microscopy. Further, we have shown that cardiac cells encapsulated using our
visible light crosslinking platform have significantly greater cell viability
compared to systems using UV light for cardiac cell encapsulation.
Future experiments will be to incorporate the primary SNS and PSNS neurons
in situ
to investigate their rate of innervation, in addition to how their spontaneous
firing will affect cardiac output. This development of a
physiologically relevant model of the cardiac ANS will enable the systematic
investigation of novel therapies to promote/prevent the intervention of
different neural populations, in addition to improving our understanding of
cardiac dysautonomia.

References:

[1] Ripplinger et al., Prog Biophys Mol Biol, 2016. 120(1-3): p. 199-209.

[2] Cao et al.,
Circ. Res., 2000. 86(7): p. 816-21.

[3] Chrousos and
Gold, JAMA, 1992. 267(9): p. 1244-1252.

[4] Ben-Zvi et
al.
, PLoS Comput. Biol., 2009. 5(1).

[5] Binkley et
al.
, JACC, 1991. 18(2): p. 464-472.

[6] Hou et al.,
Heart Rhythm, 2016. 13(2): p. 584-92.

[7] Florea and
Cohn, Circ. Res., 2014. 114(11): p. 1815-1826.

[8] Kobayashi
et al.
, Ann Thorac Surg, 2013. 96(1): p. 339-45.

[9] Triposkiadis
et al.
, J Am Coll Cardiol, 2009. 54(19): p. 1747-62.

[10] Vanoli et
al.
, Circ. Res., 1991. 68(5): p. 1471-81.

[11] Calvillo
et al.
, Journal of …, 2011.

[12] Klein and
Ferrari, Cardiology journal, 2010. 17(6): p. 638-644.

[13] Brack et
al.
, Cardiovascular Research, 2011. 91(3): p. 437-446.

[14] Oiwa et
al.
, Integr Biol (Camb), 2016. 8(3): p. 341-8.

[15] Takeuchi
et al.
, Lab Chip, 2011. 11(13): p. 2268-75.

[16] Takeuchi
et al.
, Integr Biol (Camb), 2012. 4(12): p. 1532-9.

[17] Miwa et
al.
, PLoS One, 2013. 8(7): p. e65202.

[18]
Toepke and Beebe, Lab Chip, 2006. 6(12): p. 1484-1486.