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(620bz) Investigating Neural Control of Epithelial Barrier Function of the Small Intestine with an in Vitro Engineered Model System

Authors: 
Puzan, M., Northeastern University
Koppes, A., Northeastern University

Introduction:

The epithelial cell barrier of the intestine
controls the absorption of metabolites from the intestinal lumen. Improper
function of this barrier can lead to inflammation and a ?leaky gut', where
tight junctions do not form between enterocytes and toxins or bacteria may pass
between these cells and into the body.  This
dysregulation plays a critical role in intestinal diseases such as irritable
bowel disease (IBD), which affects up to 15% of the population of the United
States (1).

Current in
vitro
models of the epithelial cell barrier typically consist of polarized epithelial
colorectal adenocarcinoma cells (Caco-2) grown on a polymer transwell
insert (2). Barrier function may be studied in various conditions based on the
tight-junction resistance and flux through the transwell
membrane. These models fail to consider the extensive neural network that lines
the gastrointestinal tract (GI), the enteric nervous system (ENS), which modulates
intestinal secretion and motility (3). Enteric glia, the support
cells of the enteric nervous system, have also been reported to impact
epithelial survival and maturation (4).  Therefore,
it is the hypothesis of this work that a co-cultured
model consisting of both Caco-2 and enteric neurons and/or glia will provide a
more realistic in vitro model of the
epithelial cell barrier and will help to elicit the role of the enteric nervous
system in barrier function regulation of healthy tissue and disease states.

Materials
and Methods:

A mixed culture of enteric neurons and glia was
isolated from 10 week old, adult black 6 mouse small intestine, (5) and seeded
onto laminin coated glass cover slips in 24 well plates.  Neurons were grown under standard incubated
culture conditions in Neurobasal media supplemented
with NGF and GDNF to promote neural survival in vitro.

At day 8 of neuron culture, Caco-2 were seeded onto 0.4 um PET transwells at approximately
60,000 cells/cm2. The neurons remained on coverslips beneath the
transwell. Control cultures were Caco-2 with no enteric cells, and enteric
cells with no Caco-2.  NGF/GDNF Supplemented
Neurobasal media was used for both cell lines. After
5 days of coculture, TEER (transepithelial
electrical resistance) measurements were taken with an EVOM from World
Precision Instruments (in PBS, 37°C), and then both neurons and Caco-2 were
fixed and stained. Enteric neurons were stained for Beta-III-tubulin to label
neurites and nuclei (DAPI); Caco-2 were stained for
ZO-1, a tight junction associated protein, and nuclei (DAPI). Cells were mounted
on glass slides and imaged using an inverted Olympus fluorescent microscope.

Results
and Discussion:

Enteric neurons and Caco-2 were cocultured with the Caco-2 in transwells and neurons on
cover slips in the bottoms of the wells. The cells lines were kept spatially
separate, but shared media and could thus interact through excreted metabolites
and soluble proteins. Preliminary results show that the addition of enteric
neurons to Caco-2 increases the transepithelial
resistance and thus tight junction formation. After 5 days of culture, TEER measurements
for the cocultured Caco-2 (Fig 1) were 270.8 +/- 3.9
ohms, while those of the Caco-2 alone were 238.6 +/- 10.0 ohms, a ~14% increase
in a short duration culture. This indicates that soluble factors released from
the enteric neurons impact the initial formation of tight junctions with this
cell line.

Figure 1: Transepithelial
Resistance (TEER) for co-cultured Caco-2 and enteric neurons is 14% higher than
the control after 5 days of culture

Conclusions:

These preliminary results imply a role of the
enteric nervous system in the regulation of epithelial barrier function and
flux across the epithelium. Further work will investigate the formation of
tight junctions with coculture for an extended length
of time (10-28 days) and determine if the addition of neurons and enteric glia
to an in vitro model impacts transepithelial flux. Additionally, the epithelial cell
line used in the coculture will be changed to include
primary small intestinal stem cells (6) and/or mucous producing cells such as
HT-29 (7) to determine if neural inclusion impacts barrier function. Neurite
extension and functionality will be examined via neural tracing software and
electrophysiology. This platform may provide mechanistic insight into the
cross-talk between the epithelium and the enteric nervous system, which may
lead to a more realistic pharmacological test bed that recapitulates the
complex biological environment of the small intestinal niche.

Acknowledgements:
The
authors thank members of the ABNEL group and Northeastern University for their
support. Authors also thank Dr. Rebecca Carrier for her insight into epithelial
model systems.

References:

1.  Quigley,
E., Fried, M., Gwee, K. A., Olano,
C., Guarner, F., & Khalif,
I. (2009). Irritable bowel syndrome: a global perspective. WGO
Practice Guideline.

2. Hidalgo, I. J., Raub,
T. J., & Borchardt, R. T. (1989). Characterization of the human colon carcinoma cell line (Caco-2) as
a model system for intestinal epithelial permeability. Gastroenterology, (96),
736-49.

3. Grundy, D., Schemann,
M., (2006) Enteric Nervous System. Curr. Opin. Gastroenterol. 22. 102-110.

4. Gulbransen B.,
Sharkey, K. (2012) Novel functional roles for enteric glia in the gastrointestinal
tract. Nat. Rev. Gastroenterol.
Hepatol
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5. Smith, TH., Ngwainmbi,
J., Grider, JR., Dewey, WL.,
Akbarali, HI. (2013). An
in-vitro preparation of isolated enteric neurons and glia from the myenteric plexus. J Vis Exp. (78).

6. Sato, T., Vries,
R. G., Snippert, H. J., van de Wetering,
M., Barker, N., Stange, D. E., ...
& Clevers, H. (2009). Single Lgr5 stem cells
build crypt villus structures in vitro without a mesenchymal niche. Nature, 459(7244), 262-265.

7. Rousset, M.
(1986). The human colon carcinoma cell lines HT-29 and Caco-2: two in vitro
models for the study of intestinal differentiation. Biochimie, 68(9), 1035-1040.