(556d) Towards Rapid Prototyping of a Patient Derived Gut-on-a-Chip

Koppes, A., Northeastern University
Hosic, S., Northeastern University
Murthy, S., Northeastern University
Breault, D., Boston Children's Hopsital
Zhou, F., Boston Children's Hopsital
Puzan, M., Northeastern University

Title: Towards Rapid Prototyping of a Patient
Derived Gut-on-a-Chip


Introduction: Micro physiological cell culture systems or
“organs-on-chips” have garnered interest from both academia and industry1. The future for
organs-on-chips is particularly exciting as integrating patient derived cells
may enable personalized medicine. Despite increased
publications describing organs-on-chips, their use is concentrated among bioengineering
research groups and chip parallelization remains challenging. Facile, rapid,
economic, and reliable fabrication of organs-on-chips would promote
interdisciplinary adoption and development. However,
the currently prevalent microfabrication of organs-on-chips via
poly(dimethylsiloxane) (PDMS) soft lithography2 may limit widespread
access to micro physiological systems. The fabrication of bilayer
organs-on-chips via PDMS soft lithography requires microfabrication training
and infrastructure. Because the initial design and prototyping phase may
require multiple iterations, lithographic mold fabrication can be prohibitively
expensive ($150-$500 per design dependent on feature resolution). Several
intrinsic material properties may limit the use of PDMS organs-on-chips toward
drug discovery. PDMS absorbs hydrophobic molecules which can alter
concentrations during drug pharmacokinetic studies. PDMS’ high gas permeability
prohibits O2 tension control for recapitulating hypoxic tissues such
as the intestine.PDMS’ high water vapor permeability results in
evaporation leading to bubble formation or high osmolarity, which can block flow
and impact cell viability. Finally, bonding PDMS to polymeric materials
requires additional processing such as silanization.

To simultaneously promote interdisciplinary adoption of
organs-on-chips and overcome the limitations of PDMS soft lithography, we
developed a “laser cut and assemble” process for manufacturing thermoplastic, membrane-integrated,
organs-on-chips. The human small intestine was first recapitulated by culturing
human colorectal adenocarcinoma derived Caco-2 cells on chip. Toward a more
physiological model, the small intestine was recapitulated using human patient
derived intestinal epithelial cells3.

Methods: A membrane integrated, bilayer organ-on-chip (Figure 1)
was fabricated using a benchtop laser engraver system and commercially
available acrylic sheets, double sided adhesives, and polycarbonate track etch
membranes. The human intestine was recapitulated by culturing colorectal
adenocarcinoma derived Caco-2 cells in the apical fluid compartment on a 1.0µm
membrane under both apical and basal culture medium perfusion. Caco-2 cells on
chip were compared to control cultures of Caco-2 cells on Transwell inserts via
immunofluorescent staining for tight junction protein, F-actin, and cell
nuclei. Mucus production on chip was compared to control Transwell cultures via
immunofluorescent staining for mucin protein MUC2 and alcian blue staining. Caco-2
cell maturation on chip was compared to control Transwell cultures via an
alkaline phosphatase (AP) assay. Primary human intestinal stem cells were
cultured and expanded as 3D organoids embedded in Matrigel and primary
intestinal monolayers were formed by dissociating organoids into single cell
suspensions and seeding Transwell inserts and bilayer chips. Primary intestinal
cells on chip were compared to control cultures on Transwell inserts via immunofluorescent
staining for tight junction protein, F-actin, and cell nuclei.

Results and Discussion: The laser cut and
assembly fabrication methodology boasts several key improvements compared to
PDMS soft lithography: chip fabrication throughput increased from days to hours
while cost remained < $2 per chip (cost of materials), the technique didn’t require
any specialized microfabrication, the thermoplastic chip construction limited
media evaporation and may enable O2 tension control, and the double
sided adhesives simultaneously provided fluidic compartments  and leak free bonding
without additional processing (Figure 1). Immunofluorescent staining
revealed the chip construction was biocompatible with human Caco-2 cells that
similarly expressed tight junctions and F-actin compared to Transwell control
cultures (Figure 2). An AP assay demonstrated that Caco-2 cells on chip
matured ~4x faster compared to cells under static culture medium on Transwell
inserts and exhibited a 2.2 fold increase in AP expression (p-value <
0.0001) compared to a Transwell model. Immunofluorescent staining and alcian
blue staining showed that Caco-2 cells on chip produced more mucus compared to
Transwell control cultures. Immunofluorescent staining revealed that patient
derived intestinal cells remain viable on chip over a 6 day period. 

Conclusions and Future Outlook: The
low cost, rapid, and facile fabrication technique presented here will enable
widespread access to micro physiological systems to researchers without
microfabrication infrastructure or training. Ongoing research is focused
on optimizing attachment conditions to generate robust patient derived
intestinal monolayers on chip and differentiation of said monolayers. Future
work will include integration of a patient derived enteric nervous system in
the basal compartment to recapitulate the enteric nervous system and epithelium
tissue interface.

Acknowledgements: The authors thank the NIH
NIBIB Trailblazer R21EB025395 for funding support. We also thank Northeastern
University and Chemical Engineering for start-up support.

Selected References:

(1) Esch, E. W.;
Bahinski, A.; Huh, D. Nat Rev Drug Discov 2015, 14 (4),

(2) Huh, D.; Kim,
H. J.; Fraser, J. P.; Shea, D. E.; Khan, M.; Bahinski, A.; Hamilton, G. A.;
Ingber, D. E. Nat Protoc 2013, 8 (11), 2135-57.

Sato, T.; Vries, R. G.; Snippert, H. J.; van de Wetering, M.; Barker, N.;
Stange, D. E.; van Es, J. H.; Abo, A.; Kujala, P.; Peters, P. J.; Clevers, H. Nature
2009, 459 (7244), 262-265.