(507g) Invited: Rapid Prototyping of Multilayered, Thermoplastic, Patient-Derived Organs-on-Chips

Title: Rapid Prototyping of Multilayered,
Thermoplastic, Patient-Derived Organs-on-Chips

Authors: Sanjin
Hosic1, Eric Stas2, David Breault2, Shashi Murthy1, Ryan Koppes1, Abigail
Koppes2 1Department of Chemical Engineering, Northeastern University. 2
Department of Pediatrics, Harvard Medical School.

Introduction:
Microphysiological 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 technological 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, can leach cyclosilane, and high gas permeability prohibits O2 tension
control for recapitulating hypoxic tissues.

To overcome the limitations of PDMS soft lithography, we
developed a “laser cut and assemble” process for manufacturing thermoplastic,
membrane-integrated, organs-on-chips. The fabrication technique was first
validated by culturing both Caco-2 and primary human organoid derived
monolayers on a traditional bilayer organ chip. Next, the fabrication technique
was used to develop a tri-layer organ chip integrating both 2D monolayer and 3D
culture of primary human intestinal epithelium.

Methods: A
membrane integrated, bilayer organ-on-chip (Figure 1a) 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. Next, a dual membrane integrated, trilayer organ-on-chip (Figure 1b) was fabricated to integrate primary intestinal
monolayers and intact organoids. Monolayer and organoid morphology was
visualized via confocal microscopy and cell nuclei staining.

Results and
Discussion: The technique presented here rapidly (hours) produced
inexpensive (~$2 per chip, cost of materials) organ chips using only a benchtop
laser. CAD-based manufacturing enabled iterative design with zero tooling or
mold costs. The double sided adhesives simultaneously
provided fluidic compartments and leak free bonding without additional
processing. Collectively, the technique presented here had significant
advantages in cost, throughput, scalability, and equipment requirements as
compared to PDMS soft lithography. Furthermore, the thermoplastic chip
construction limited media evaporation, may enable O2 tension control, and may
prevent absorption of hydrophobic compounds in future studies.
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. An AP assay
demonstrated that Caco-2 cells on chip matured ~4x faster compared to cells
under static culture medium on Transwells, exhibiting
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. Immunostaining revealed that
patient derived intestinal monolayers and organoids remain viable on chip for
up to 10 days. Primary monolayers exhibited 3D morphology spanning 100-200 µm (Figure 2a), expressed tight junctions
and F-actin similar to Transwell controls, and
organoids expressed the proliferation marker Ki-67 (Figure 2b).

Conclusions and
Future Outlook: The many features of cut and assemble chips, including the
low gas and water vapor permeability of thermoplastics, compared to PDMS, the
rapid, easy, and economical fabrication method as well as the ability to make
custom multilayered chips, make cut and assemble fabrication well suited for
wider adoption and development of organs-on-chips. Ongoing work towards
integration of a patient derived enteric neurons and glia to recapitulate the
enteric nervous system and epithelium tissue interface as part of a model of
the brain-gut-axis, as well as on chip instrumentation for live read-outs of cellular
activity is underway.

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), 248-60.
(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.