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GUT-ON-CHIP: HOW TO RECAPITULATE PHYSICAL AND
BIOLOGICAL FEATURES IN VITRO
Marine Verhulsel1, Anthony Simon2, Davide Ferraro1, Cécile Bureau1, Jean-Louis Viovy1,
Danijela Vignjevic2 & Stéphanie Descroix1
1
Institut Curie, PSL Research University, CNRS, UPMC, UMR 168, 26 Rue d’ Ulm, 75005, Paris (France)
Institut Curie, PSL Research University, CNRS, UPMC, UMR 144, 26 Rue d’ Ulm, 75005, Paris (France)
2
ABSTRACT
This paper describes a robust in vitro model which faithfully recreates the anatomy of small intestine on
a chip. We used microfabrication and hydrogel molding to replicate the main characteristics of the
intestine, namely its 3D structure and composition of its extracellular matrix (ECM). In particular,
biomimetic hydrogel based on collagen was molded to reproducing the intestine topography. On this
scaffold, we succeed to grow a primary epithelium, which makes this system a relevant model to study
intestine physiology.
KEYWORDS: Intestine, hydrogel, Extracellular matrix, primary cells
INTRODUCTION
The small intestine achieves most of the nutrient absorption due to its characteristic morphology: a
defined succession of villi and crypts that considerably increases the exchange area (human intestine
presents a surface area of 300m2) . More in details, the intestinal epithelium consists of a cellular
monolayer and exhibits a characteristic morphology made of villi surrounded by 5-10 crypts. Villi are
finger-like structures that rise into intestinal lumen. The high renewal rate of its epithelium
(approximately 4-5 days [1]) is very specific to this organ. The separation of intestinal functions into
dedicated morphological subunits (proliferative cells being restricted to the crypts and
differentiated/absorptive cells to the villi), makes the intestine a very attractive organ for studying the
complex mechanisms that drive epithelial dynamics.
In vivo studies of the intestinal epithelium allow limited control of physical, chemical and biological
parameters and are challenging for imaging. Clevers’ group developed an in vitro assay in which
intestinal stem cells, when seeded in a biomimetic hydrogel, spontaneously grew into organoids.
Organoids are cysts with budding structures that resemble crypts but fail to reproduce villi [2]. Two
groups tried to overcome this problem using microfluidics. Ingber’s team suggested that peristaltic
motion could induce villi formation. Nevertheless the cell line they seeded on a porous membrane
undergoing peristaltic motion only showed poor spatial organization with regards to the actual
topography of the villi [3]. March’s lab succeeded to grow primary cells on microstructured villi which
were made of non-physiological polymer and therefore lack cell-matrix interactions present in vivo [4].
EXPERIMENTAL
We propose a new device that recapitulates the biological and physical features of the in vivo
intestine. By combining micromilling (Figure 1) we obtained a brass mold that could mimic roughly
intestine 3D structure (Figure 1) and combined with soft lithography techniques, we have molded
collagen I into 3D sinusoidal structure of 400µm in height with a 400µm period (figure 2). We have
investigating different coatings strategies based on Matrigel or laminin as coating agents to reproduce the
basement membrane.
978-0-9798064-8-3/µTAS 2015/$20©15CBMS-0001
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19th International Conference on Miniaturized
Systems for Chemistry and Life Sciences
October 25-29, 2015, Gyeongju, KOREA
Figure 1 : (i)&(ii) SEM images of the brass mold. (iii)Principle of collagen scaffold fabrication.
RESULTS AND DISCUSSION
We first demonstrate the biocompatibility of our biomimetic scaffold. Mixing fibroblasts with collagen
prior to collagen polymerization allowed their seeding inside the matrix where they actually belong in vivo (Figure 2). Fibroblasts, together with epithelial cells, synthesize a specialized network of ECM proteins, basement membrane (BM), which separates epithelium from stroma. Since BM is essential for epithelial survival and growth, we successfully coated the collagen structure with thin and homogeneous
layer of laminin, a major constituent of BM. On those structures, we have then seeded early-stage cystlike organoids which opened up, spread and successfully colonized the whole structure forming a confluent epithelium monolayer (Figure 3). The labeling of proliferative cells confirmed their location in the
crypt as observed in vivo.
Figure 2 : 2D projection of fibroblasts seeded in the microstructured collagen matrix. (Fibroblast
nuclei labeled in red, collagen fibers in blue and green). Higher color intensity correlates to higher
density of collagen fibers. Therefore the 4 spots represent the raised areas of the collagen mold
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Figure 3: After 7 days the primary epithelial monolayer colonized the whole structure.(nuclei in blue,
actin in green).
CONCLUSION
Our platform provides the first primary intestinal tissue with physiologically relevant geometries and microenvironment. This realistic in vitro model opens new opportunities to elucidate the main parameters
driving cell proliferation, migration and differentiation that are difficult to assess in vivo. In addition, it
will allow us to investigate the effects of geometry, stiffness of the matrix or role of fibroblasts in those
processes.
ACKNOWLEDGEMENTS
The authors acknowledge EU funding (ERC CellO)
REFERENCES
[1] Van der Flier L. G., & Clevers H. (2009). Stem cells, self-renewal, and differentiation in the intestinal epithelium. Annual review of physiology,71: 241–60.
[2] Sato T et al (2009) Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature, 459(7244), 262-5.
[3] Kim HJ et al(2013) Human gut-on-a-chip inhabited by microbial flora that experiences intestinal
peristalsis-like motions and flow. Lab chip, 12 (12), 2165-74
[4] Costello CM et al (2014) Synthetic small intestinal scaffolds for improved studies of intestinal differentiation. Biotechnol Bioeng, 111(6):1222-32
CONTACT
* Marine Verhulsel : [email protected]
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