Engineering tools for providing mechanical cues to intestinal in vitro models

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UNIVERSITÁ DI PISA

DOTTORATO DI RICERCA IN INGEGNERIA DELL’INFORMAZIONE

Engineering tools for providing

mechanical cues to intestinal in vitro

models

DOCTORAL THESIS

Author Joana Marques e Costa

Tutor (s)

Prof. Arti Ahluwalia Prof. Claudio Domenici Dr. Tommaso Sbrana Reviewer (s)

Prof. Federico Carpi Prof. James Busfield

The Coordinator of the PhD Program Prof. Marco Luise

Pisa, November 2018

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This thesis is dedicated to Avó Belmira, a true badass

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“We especially need imagination in science. It is not all mathematics, nor all logic, but it is somewhat beauty and poetry.”

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Acknowledgements

IRST of all I would like to thank my three supervisors who gave me the opportunity to pursue my PhD studies and are responsible for the success of this work. I express my earnest gratitude to Professor Arti Ahluwalia for hosting me in her research group, providing me guidance, support, inspiration, and for allowing me to learn so many new things and to grow as a researcher. I am deeply grateful to Tommaso Sbrana, for kindly integrating me not only in the MICACT network, but especially in the IVTech team, dedicating his time to guide me and to help me growing professionally. I wish to acknowledge Professor Claudio Domenici for generously hosting me at CNR being always welcoming and flexible to help me developing my work.

Since my research could not happened without financial support, I would also like to thank IVTech and the MICACT network for the funding.

I also would like to express my deepest appreciation to Daniele Cei, who was a great tutor. His infinite kindness and ability to teach me were very important for this work and he should win the Nobel prize of Patience.

I would like to thank my colleagues of the IVM group: Ludo, my fearless companion in all the PhD ‘battles’ - it was a pleasure to go through this adventure together with her (with the aid of a calculator, of course); Chiara, aka Pasqua, who is an untamed fox but somehow was always by my side; Robi, with whom I like to talk about whatever subject because she is an automatic joy transmitter; Giorgio, the incredible wizard of all the complete answers on any subject; Alejandro, who must give me recipe for being such a nerd and cool guy at the same time; and Daniele, who could sustain an entire city if we could convert his focusing power in electricity. I also want to thank some people that are not currently part of our research group but were crucial for my early times as a PhD student: Serena Giusti, Daniela Giacopelli and Giulia Gori.

The guidance I received was not provided only by the IVM group colleagues, it often resulted from a spontaneous “I can help you to do that” from someone in the office/biofabrication lab/corridor/every place at Centro Piaggio. That is why I wish to sincerely acknowledge Francesca, Carmelo, Professor Vozzi, Aurora and Salvatore. I thank also all the other “bio guys”: Lorenzo, Roberto, Anna, Licia and Luigi (aka Gigi), for contributing for such cool working environment.

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I also want to thank the students I followed in these three years: Daniele Dinelli, Marta Feula, Marta Macchi, Roberta Irardi, Joana Dias and Vanessa Almonti. They all helped me and taught me something in one way or another, because tutoring is also a learning experience.

To wrap up, (and because I might be forgetting someone), I am grateful to all people working at Centro Piaggio, who hosted me so affectionately - without their precious support this journey would have not been the same.

Since my work was also developed in collaborations with other labs and institutions, my special thanks are extended to Professor Federico Carpi from UNIFI, Alessandro Corti from the Pathology Department of St. Chiara Hospital, and Federico Vozzi from CNR. Naturally, I also express by gratitude to Professor James Busfield and the Soft Matters group, from the SEMS department of the QMUL, for receiving me so well in my period abroad. I am particularly grateful to Michele, a unique species from the research underworld that metabolizes science into enthusiasm, with whom I developed part of my project.

Once my PhD studies implied finding in Pisa my second home, I would also like to thank some other people that contributed for that to happen. I will start with my housemate Chiara that is way more than that, she is my italian sister. Then of course, I thank my previous housemates: Elisa (aka Annalise), Federica and Anna; and the other “pisani” friends: Sariah, Ale, Dani, Antonio, Giamma and Carla. I am especially grateful to Danilo, who told me that being “scalable” is a good property of a product, and he was right: in a couple of years a prototype can be scaled-up into a very good product.

Because the support of my long-time friends has always been crucial for my accomplishments, I thank my dear friends from home: “a malta de Sever” and my “Tecos do Porto”.

Finally, the deepest acknowledgement goes to all my loving family, especially my parents, sister and nephew. As eloquent I could be, it would always be superfluous to express my gratitude to them in some words, so I will just leave my “Obrigada Mamã, Papá, Diana e Miguel”.

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Summary

SING in vitro cell cultures to study organ-level behavior is a directly scalable and

robust approach, but its predictive power is limited by the lack of biological functionality. On the other hand, animal models replicate organ- and multi-organ-level function but are intrinsically defective due to undeniable differences between animal and human physiology. Moreover, they are more expensive than in vitro models and raise ethical issues. There is then the need to develop in vitro models that can provide robust data and, at the same time, replicate organ-level function, obtaining in this way accurately predictive results.

This thesis aimed at improving the current intestinal in vitro models, by addressing the physiologic mechanical cues that are missing in traditional static 2D models: a realistic topography of the intestine, a flow of nutrients and oxygen, and the motility of the intestine walls. The PhD project was carried out in the context of a Marie Skłodowska-Curie ITN on electroactive polymers (grant agreement No 641822 "Microactuators - MICACT), thus much of the work was focused on the applications of dielectric actuators for realizing models of intestinal peristalsis.

To mimic the architecture of the intestinal villi, a 3D scaffold was fabricated. Poly(lactic-co-glycolic acid) was cast into molds (produced from rapid prototyping methods) and, by employing techniques of thermal induced separation and porogen leaching, porous biocompatible scaffolds were obtained from it. Data from imaging, cell culture and permeability tests confirmed the suitability of these structures to mimic the topography of the intestinal epithelium and to support the culture of intestinal cells. Additionally, the scaffolds were integrated in a dual flow bioreactor and showed that their integrity was maintained during exposure to constant flow for 3 weeks, opening the possibility to include them for an in vitro model where the presence of an adequate flow of nutrients and oxygen is provided to the 3D cell construct.

To mimic the motility of the intestinal walls, electroactive polymers were used to construct a stretchable substrate for cells. Progressively refining the characteristics of the produced devices, three bioreactors prototypes were developed. As main outcomes, a planar actuator capable of providing 10% of radial in-plane deformation to Caco-2 cells was established,

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demonstrating the suitability of the Dielectric Elastomer Actuators (DEA) technology for biomimetic muscle-like actuation. Moreover, after changing the configuration of the DEA actuator, around 5% of equiaxial strain was provided to cultured fibroblasts, inducing changes in the organization of the cell cytoskeleton.

The research presented in this thesis illustrates the great potential of exploring both electroactive polymers and microfabrication technologies for the development of biomimetic bioreactors. The outputs of this work can overcome some of the shortcomings of traditional in vitro models and help forging the path towards “ideal” in vitro models which integrate all the cues present in the biological milieu.

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List of publications

International Journals

1. L. Ricotti, G. Gori, D. Cei, J. Costa, G. Signore, and A. Ahluwalia, “Polymeric Microporous Nanofilms as Smart Platforms for in vitro Assessment of Nanoparticle Translocation and Caco-2 Cell Culture,” IEEE Transactions on NanoBioscience, vol. 15, no. 7. pp. 689–696, 2016.

2. D. Cei, J. Costa, G. Gori, G. Frediani, C. Domenici, F. Carpi, and A. Ahluwalia, “A bioreactor with an electro-responsive elastomeric membrane for mimicking intestinal peristalsis.,” Bioinspir. Biomim., vol. 12, no. 1, p. 16001, 2016.

3. J. Costa, A. Ahluwalia, “A guide for intestinal in vitro models: advances and current challenges”, SUBMITTING

4. J. Costa, R. Irardi, M. Macchi, and A. Ahluwalia, “Development of a porous scaffold to mimic the topography of the intestinal epithelium”, IN PREPARATION

5. J. Costa, M. Ghilardi, H. Boys, J.J.C. Busfield, A. Ahluwalia, F. Carpi, “Development of an innovative bioreactor based on dielectric elastomer actuation for mechanical stimulation of cells in vitro”, IN PREPARATION

6. L. Cacopardo, J. Costa, S. Giusti, L. Buoncompagni, S. Meucci, A. Corti and A. Ahluwalia, “A Smart Bioreactor for Cellular Impedance Monitoring and Real Time Imaging of Biological Barriers”, IN PREPARATION

International Conferences/Workshops with Peer Review

1. J. Costa, D. Cei, C. Domenici, F. Carpi, A. Ahluwalia, “Development of a new electrically driven moving interface for the representation of the permeable intestinal mucosa”, Advances in Cell and Tissue Culture, Barcelona, Spain, 30 May-1 June 2016 (poster)

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2. J. Costa, D. Cei, E. Vanello, A. Ahluwalia, “Development and optimization of a moving interface for in vitro models of epithelial barriers using an EAP”, 6th International conference on Electromechanically Active Polymer transducers & Artificial Muscles, Helsingør, Denmark, 14-15 June 2016 (poster)

3. J. Costa, D. Cei, E. Vanello, D. Dinelli, C. Domenici, A. Ahluwalia. “Development of an innovative moving interface for an in vitro model of the epithelial intestinal barrier”, European Congresses on Alternatives to Animal Testing, Linz, Austria, 24-27 August 2016 (oral presentation)

4. J. Costa, M. Feula, D. Cei, A. Ahluwalia, “A bioreactor using a porous EAP actuator as a physiological-like interface for cell culture studies”, 7th International conference on Electromechanical Active Polymer transducers & Artificial Muscles, Cartagena, Spain, 6-7 June 2017 (poster)

5. J. Costa, M. Ghilardi, H. Boys, J. J.C. Busfield, A. Ahluwalia, F. Carpi, “Innovative bioreactor based on dielectric elastomer actuation to dynamically stretch cells in vitro”, 8th International conference on Electromechanical Active Polymer transducers & Artificial Muscles, Lyon, France, 5-6 June 2018 (poster)

6. J. Costa, R. Irardi, M. Macchi, A. Ahluwalia, “Development of a porous scaffold to mimic the topography of the intestinal epithelium”, Sixth National Congress of Bioengineering, Milan, Italy, 25-27 June 2018 (oral presentation).

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List of Abbreviations

B

BSA Bovine serum albumin

D

DAPI 4′,6-diamidino-2-phenylindole

DEA Dielectric elastomer actuator

E

EAP Electroactive polymers

ECM Extracellular matrix

EVA Ethylene-vinyl acetate

G

GIT Gastrointestinal tract

H

HDPE High-density polyethylene

F

FBS Fetal bovine serum

FITC Fluorescein isothiocyanate

FPS Frame per second

L

LDPE Low-density polyethylene

M

M (cell) Microfold cell MLC Myosin light chain

P

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viii PES Polyethersulfone

PET Polyethylene terephthalate

PFA Paraformaldehyde

PKS Protein kinase C

PLGA Poly(lactic-co-glycolic acid)

PS Polystyrene

PU Polyurethane

PVA Polyvinyl alcohol

S

SEM Scanning electron microscope SIS Small intestinal submucosa

T

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CHAPTER

Motivation and Outline

This study was performed in the framework of the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No 641822 "Microactuators – MICACT”. Precisely, my work was sponsored by the project partner IVTech, a startup from the University of Pisa whose goal is to provide in vitro technologies for the development of physiologically relevant tissue models. The aim was to develop biomedical applications of Electroactive Polymers, which are novel materials with actuation capabilities. In this context, my project was focused on an in vitro intestinal model which promotes possible alternatives to animal experimentation.

Therefore, this work has been developed following the 3R’s principles: ‘Replacement, Reduction and Refinement’ as laid out by Directive 2010/63/EU on the protection of animals used for scientific purposes. It foresees to ‘Replace’ animals used in experiments with non-sentient alternatives; to ‘Reduce’ the number of animals employed; and to ‘Refine’ animal experiments so that they cause minimum pain and distress. The practice is embraced not only by those who are against animal experimentation, but also by those who perform experiments on animals: even in the cases when in vivo experimentation is claimed to be indispensable, scientists agree on the need to improve animal welfare [1]. Currently, most of the studies in life sciences are performed on traditional simple two-dimensional (2D) static human in vitro cell cultures, or on rodents (mentioning the most used animal model [2]). Besides the ethical issues, attending the ‘Replacement’ principle can generate some technical debate: while the approach to study organ-level behavior in cell culture is directly scalable and robust, its predictive power is limited by the lack of

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biological functionality and it is, indeed, a reductionist tactic. On the other hand, animal models replicate organ- and multi-organ-level function, but are intrinsically defective due to undeniable differences between animal and human physiology and, moreover, are more expensive than in vitro models [3]. Hence, the success key relies on developing in vitro models that can provide robust data, replicating at the same time organ-level function and dynamics, obtaining in this way accurately predictive results.

My research has been directed to apply this solution to model the human intestine – the vital organ of the gastrointestinal tract where theabsorption of nutrients and several other phenomena take place. The goal of improving the traditional in vitro models, by creating an innovative and accurate cell culture system, foresees collective efforts and investigation in different research fields. With plenty of scientific aspects still to be explored, I have so far addressed some of the ‘fundamental engineering issues’ that must be resolved to obtain the “ideal” intestinal in vitro model.

As a first step, I have identified the physiologic key elements that are missing in traditional static 2D models: the presence of a realistic topography of the intestine (an epithelium folding in villi); the presence of the flow of nutrients, as it happens in the human intestine upon digestion; and the motility of the intestine walls. They are part of the crucial components of the intestinal epithelium microenvironment represented in Figure 1.

Figure 1 - Components that represent the intestinal epithelium microenvironment

Each of the referred element concerns a specific kind of mechanical cue, and for each one I have developed an ‘in vitro tool’ to provide the necessary mechanical input. Those aspects were addressed individually as a way of dividing and organizing my work. Reflecting this vision, I have dedicated different chapters of this thesis for each of the mechanical cues. In each section, the key element is addressed, as well as the technologies I used to develop the respective in vitro tool.

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The introduction to the present thesis composes Chapter 2, where the characteristics of the organ under investigation relevant for this work are presented. Chapter 3 presents the state of art of the in vitro methods that are used to model the intestine. Chapter 4 is dedicated to the elaboration of a cell substrate that resembles the topography of the intestine. The main techniques of microfabrication of porous 3D scaffolds for cell culture are reviewed and the obtained results are presented. The compatibility of the developed scaffold with a flow system bioreactor is assessed here as well. Then, Chapter 5 explores the topic of mechanical stimulation of cells. The technology I used to address this issue – electroactive polymers – is examined, and the results that consist in the production of three bioreactor prototypes are presented and discussed. The overall goal of my research is to finally merge together each developed tool in one single in vitro model, and such possibility is discussed in Chapter 6 after presenting the main conclusions of the present work.

To better elucidate the outline of the thesis, Figure 2 illustrates the division of its contents by theme and respective technology/ in vitro tool.

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CHAPTER

Introduction

2.1 The human intestine

The intestine is the organ responsible for the digestion and absorption of nutrients, but it also has secretory and immune functions [4].

The small intestine is the longest part of the digestive tract and is divided in three portions: the duodenum, the jejunum, and the ileum [5]. In turn, the wall of the small intestine is divided into four layers that are connected by connective tissue and neural and vascular components: the mucosa, the submucosa, the muscularis propria, and the serosa [4], as presented in Figure 3.

Figure 3 – Representation of the different layers of the small intestine, adapted from [4].

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The intestinal epithelium, the most external layer of the mucosa, is the most self-renewing tissue of adult mammals and it is represented in detail in Figure 3 [6]. It is composed of different cell types, each one specialized in a different function: enteroendocrine cells, Paneth cells, goblet cells, enterocytes, and microfold (M) cells [6]. The enteroendocrine cells coordinate gut functioning through specific hormonal secretion. Paneth cells reside at the crypts and have a role in innate immunity by secreting proteins such as antimicrobials, while Goblet cells, usually interspersed among enterocytes, produce and secrete mucus required for the chemical and mechanical protection of the gut. On its turn, enterocytes represent the absorptive lineage. They are columnar cells, highly polarized, with an apical brush border that absorbs nutrients and substances across the epithelium - they make up more than 80% of all intestinal cells [7]. Finally, M cells perform luminal antigen sampling so that cells of the immune system contact with potential pathogens [8].

Figure 4 - Representation of the intestinal epithelium, adapted from [9].

2.2 Intestinal villi

90% of the absorption sites in the gastro intestinal tract (GIT) occurs in the small intestine [10], since its epithelium folds into microscopic highly vascular finger-like projections, called villi. They extend from the mucosa surface to between 0.5 and 1.5 millimeters in height. Increasing furthermore the surface area, each intestinal epithelial cell presents on its apical surface roughly 1000 microvilli (around 1 micrometer long and 0.1 micrometer in diameter) that form the brush border [4,5].

Together, these structures cause an outstanding increase in the total surface area available for the absorption of nutrients – of around 600-fold [10]. Figure 5 illustrates the configuration of an intestinal villus.

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Figure 5 - Structure of an intestinal villus, adapted from [11].

Besides providing an increase in the area available for the absorption of nutrients, the villi architecture is involved in the arrangement of the different cells that compose the intestinal epithelium. In this tissue, stem cells lying in the crypts divide and give rise to committed cells that undergo through a differentiating process to form mature absorptive enterocytes. Upon reaching the crypt-villus junction, enterocytes then migrate up the villus and finally settle on the tip of the villus [12]. The mechanism by which the cells perform this migration remains still unclear, but it seems that the structure of the villi is important for enterocytes cell fate. In another study, enterocytes showed a gene-expression signature varying across the villus length, suggesting spatial heterogeneity of enterocytes along the intestinal villi [13]. These facts indicate a structural role of the villi on the development and renewal of the epithelium.

2.3 Intestinal motility

Digestive and absorptive functions of the gut are dependent on the motility of various parts of the bowel. The movements have important functional roles, including mixing, propulsion, and separation of luminal contents. These actions are possible because of the coordinated interaction of excitatory and inhibitory neurons of the enteric nervous system [14].

Specifically, the small intestine undergoes both segmental and peristaltic contractions induced by actuation of the circular and longitudinal, respectively, muscular layers of the bowel, as it is illustrated in Figure 6. Segmental concentric contractions aid in mixing and absorption of the digestion contents, by dividing the intestine into spaced segments. On the other hand, peristaltic contractions, performed by longitudinal muscles, drive the products through the intestinal tract [15]. Not only do these contractions play a fundamental role in the digestion and absorption of nutrients, they are also crucial for the proliferation and differentiation of the epithelial interface [16]. Typical values for frequency and strain reported for the intestine are 7-20 contractions per minute and

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8-7 10%, respectively [17].

Figure 6 - Different types of contractions of the intestine associated with particular functions, adapted from [14].

In addition, the intestinal villi exhibit piston-like contraction and relaxation movements, which are thought to facilitate the removal of some of the digestion products from the lymphatic vessels that course through the villi to the lymphatic system. Strands of smooth muscle within the lamina propria are thought to give rise to these movements. The villi also show swaying movements that may contribute to the mixing of digestion contents within the intestinal lumen [18].

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State of art: modelling the intestine in vitro

The study of intestinal phenomena, as well as intestinal disorders, have been empowered by using animal models, however many intestinal processes are difficult to control using in vivo models. In vitro models are widely used to simplify the study of complex phenomena of the in vivo environment, creating well-controlled and easily reachable conditions for the quantitative and repeatable evaluation of cell response. They can be used in many different fields thanks to their wide areas of application including toxicology, drug testing, tissue engineering and nutraceutics [19].

Moreover, in vitro intestinal models can potentially enable improved studies of cellular growth and proliferation, drug absorption, and host-microbial interactions, while reducing the expense and ethical issues raised by using numerous live animals [20].

3.1 Two-dimensional (2D) cellular in vitro models

One of the most common systems to re-create in vitro the intestinal interface is the Transwell®, represented in Figure 7. It has been used to investigate intestinal lumen-to-blood permeability of drugs, toxins or microorganisms. Here, the epithelial cells are seeded on the membrane that separates the apical from the basolateral compartment, corresponding respectively to the intestinal lumen and the blood vessels. Thus this system mimics to a certain extension the configuration of the in vivo intestine [21].

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Figure 7 - Representation of an insert of the Transwell® system, taken from [22].

Caco-2 cells are an immortal human cell line derived from a human colorectal adenocarcinoma that is regularly used in the models of the intestinal epithelium. When in culture, they grow into a confluent monolayer, differentiate and behave similarly to enterocytes, resembling normal intestinal epithelium [23]. It is the most widely used cell model to study the permeability of drugs over the last 20 years and it has been accepted as standard protocol to predict drug intestinal permeability in humans by pharmaceutical companies and controlling authorities [24].

The cell differentiation starts when the cells achieve confluence, around 7 days, and is completed within 21 days. That is when the cells are polarized and connected to each other through tight junctions (TJ), exhibiting an apical brush border structure with several enzymes, transporters and receptors [25].

Additionally, the Caco-2 monoculture does not contemplate some other important factors that influence the functionality of enterocytes such as the mucus layer or the interactions between the epithelium and the stroma, [26], pushing scientists to develop more complex cellular models, as presented as follows. Furthermore, in another perspective, the referred cell culture process requires about 3 weeks, which can be labor intensive and time consuming, limiting its wide application in high-throughput screening of new compounds [27]. Answering to this, some groups developed modified Caco-2 culture techniques that require a minor culturing period [28–30].

Coming as an improvement, a cell model based on the mixture of Caco-2 and mucus producing HT29 cell lines was developed. It mimics both enterocytes and goblet cells, and it was reported to generate more predictable experimental results, counting with the influence of mucus upon the transport of drugs [21,31,32]. Other models were developed to include the presence of M-cell-like cells that resemble functionally and morphologically the intestinal M cell - characterized by the sparse and irregular microvilli and a high transcytotic activity [8]. It was described that Raji B cells, induced M cell’s phenotype in some Caco-2 cells [33–36]. The rationale behind these two previous models were merged to create in a triple co-culture model with the three cell types: Caco-2, HT29-MTX and Raji B cells, was developed by Sarmento’s group [30–33].

3.2 Three-dimensional (3D) cellular in vitro models

Most of the current understanding of several biological processes are based on two-dimensional (2D) surfaces however cells in vivo exist in a heterogeneous and information-rich environment which modulates cellular events. The complex array of biochemical signals is controlled by the different cells as they secrete and absorb molecules to/from

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each other and to/from the extracellular environment. 2D models lack the representation of these interactions and are a reductionist approach that not faithfully captures the in vivo scenario. Therefore, 3D models are believed to potentially bridge the gap between cells cultures and animal models [19,39].

Regarding the importance of matrix dimensionality and the current shift from 2D to 3D models, more recent in vitro intestinal models have been focused on mimicking the intestinal mucosa that includes a broad range of stromal cells. Considering the crucial role of stromal cells and extracellular matrix (ECM) in the maintenance of intestinal epithelial cells [40], a couple of 3D model were recently purposed comprising Caco-2 and goblet-induced HT29-MTX cells and stromal cells [26,41] that better recreate the composition of the tissue.

From another point of view, the organization of the different cells into a functional 3D structure should occur spontaneously. Ideally, an in vitro model that closely resembles the human intestinal epithelium should comprise a combination of the different GI epithelial cells, obtained from an individual, that should be cultured indefinitely. In fact, the problem with primary cell cultures is that these intestinal tissue pieces could not be maintained for culture for a long period [42]. The identification of intestinal stem cells - the Lgr5 stem cells of the small intestinal and colonic crypts - came to solve this problem [43]. Since these cells can differentiate into all intestinal epithelial cells (including also stem and progenitor cells), they can be grown in vitro, for longer periods, forming the so called ‘mini guts’, or organoids [44,45]. In Figure 8 the organization of an intestinal organoid is represented. The obvious potential of these now self-sustaining organoids in the field of regenerative medicine was instantly clear, and these ‘mini guts’ were already successfully tested for engraftment in mice [46,47]. The use of organoids is a fast growing field and it has been explored for other innumerous application ranging from regenerative medicine [48] to host-microbe interaction studies [49] or drug testing and disease modelling [50]. Nevertheless, organoids also have their shortcomings. Specifically, by themselves they are unable to mimic biomechanical forces that stem cells encounter in

vivo. Furthermore, since they are heterogeneous in terms of viability, size and shape, and

their spatial arrangement limits drug penetration, they are difficult to be used for drug screens [51,52].

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3.3 The importance of providing mechanical cues

Nowadays it is widely accepted that cell differentiation and proliferation are dictated by a combination of not only chemical, but also mechanical cues. Experimental studies have demonstrated that mechanical factors, including substrate stiffness, nanotopography of the adhesion surface, mechanical forces, fluid flow and cell colony sizes can guide stem cell fate [53]. Likewise, the mechanical cues to which the intestinal epithelium is exposed should not be disregarded. Actually, epithelial cells have exceptional interactions with their microenvironment since they maintain three distinct types of interfaces in their cell surfaces: the apical surface of the epithelium is free of adhesion contact, while interacting with the dynamic external environment; the lateral surfaces interact with neighboring cells through adhesions; the basal surface of the epithelium, on the other hand, interacts with a specialized ECM. Thanks to these different interactions, biochemical and biomechanical cues regulate epithelial cell fate and even contribute to pathological processes [54–57]. Indeed, most of adult human cancers have origin on epithelial cells, and mechanical changes have been associated with epithelial carcinomas, including elevated ECM stiffness and increased interstitial pressure [58].

This kind of observations raise a lot of questions, starting with the most essential ones: how does a cell ‘feel’ and responds to the mechanical stimuli? And who are the cellular players? At the biological level exist a number of molecular mechanisms through which cells sense and transduce mechanical cues. These are localized within the membrane, the cytoskeleton and at specific cell-matrix complexes [58]. This phenomenon is defined as mechanotransduction - how cells sense physical forces and translate them into biological responses [59]. Some of these mechanisms are very similar across all domains of life and some other are more specific to a given subset of cells. As an example, the role of the mammalian cell cytoskeleton in responding to physical cues is one of the most studied examples of mechanotransduction, and only a few processes of this complex event have been elucidated. Some mechanisms illustrate direct coupling between chemical signaling and mechanical forces, such as the stretch of some molecules of focal adhesions that exposes either binding sites or phosphorylation sites, and thus trigger signaling pathways that will results in a gene expression/repression [59]. Other mechanisms present responses to physical forces that allow for adaptation of a cell and its cytoskeletal network to external changes of stiffness in less than 100 milliseconds [60]. In Figure 9 there is a

representation of some possible molecular interactions associated with

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Figure 9 - Molecular cell players and signaling pathways involved in mechanotransduction, adapted from [61].

The cell adhesion proteins - integrins and cadherins - seem to be key players in mechanotransduction. They act as receptors that transmit mechanical forces and regulate several intracellular signaling pathways. Integrins have been involved in a remarkable range of sensing and translating mechanical cues, including cellular responses to stretch, elevated hydrostatic pressure, fluid shear stress, and osmotic forces. In the first case, cells on elastic substrates are subject to strains delivered through their adhesive contacts. In other systems, the requirement for integrins in force transmission is not obvious but evidence still suggests they play an indirect role [62]. Although forming adhesions weaker than those of the integrins, some studies point out that cadherins present in cell–cell junctions show analogous force-dependent behavior. When cells expressing E-cadherin contacted with each another, it was verified an increase in the activation of the Rho protein and an increase in the phosphorylation of the myosin II regulatory light chain (MLC) indicating cell response [62].

It is then essential to provide different mechanical stimuli to in vitro cell culture to understand the molecular mechanisms regulating mechanotransduction.

Scrutinizing the organ of study - the intestine - by examining the dynamic conditions to which the epithelial intestinal cells are exposed, one can identify the main mechanical cues that should be comprised in an intestinal in vitro model. These are:

- villi topography;

- the mechanical stimulation provided by the

o intestinal motility (providing tensile and compression forces);

o and flow of medium (guaranteeing shear stress, as well as physiological-like diffusion of oxygen and nutrients).

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3.4 Intestinal topography – engineered scaffolds

The importance of the organization of cultured cells in functional 3D structures was already highlighted, and it is now important to elucidate the link between 3D structures and mechanotransduction. To study these phenomena, scientists have been developing engineered structures that ‘aid’ cells organizing themselves similarly to the in vivo microenvironment. Such structures belong to the category of cell scaffolds.

Trying to recreate the 3D architecture of the intestinal epithelium has been the goal of innumerous research groups, and some of them focused on the reconstruction of the villi architecture. It is to note that some scaffolds are developed using ex vivo explants or in

vivo-derived materials [63]. Due to the scope of the research behind this thesis, special

emphasis will be given to scaffold fabricated synthetically.

In 2011, Sung et al. [64] developed a 3D villi model consisting of a collagen hydrogel scaffold. Indeed, producing soft hydrogels in the desired shapes and dimensions using conventional techniques can be a manufacturing challenge. The authors obtained the scaffold by a combination of laser ablation and sacrificial molding technique, using calcium alginate, which allowed the maintenance of both the complexity and integrity of the hydrogel structure. Evident morphological similarities were observed between the collagen scaffolds covered with the Caco-2 cells and the human jejunal villi [64]. Later, to evaluate the integrity and the role of the 3D model in predicting drug permeability, the same group adapted the collagen scaffold to an insert design. Culturing Caco-2 cells on the scaffolds for 21 days did not only shorten the height of the villi, but also resulted in the formation of multiple layers due to cell penetration in the matrix as the collagen degraded. This led to a higher permeability of the tested hydrophilic drug, when compared with the data from 2D cultures, approximating the values obtained for the mammalian intestines. Moreover, it was observed that the cell differentiation on the 3D scaffold varied along the villus [65].

Mimicking also the villi architecture, Costello et al. [66] developed a synthetic 3D scaffold that could support the coculture of epithelial cell types with selected bacterial populations. The study aimed at exploring microbe-induced intestinal disorders and develop targeted probiotic therapies. Biodegradable and biocompatible villous scaffolds were fabricated using poly lactic-glycolic acid (PLGA), cultured with Caco-2 cells and finally used as a platform to mimic the adhesion, invasion profiles and interactions of certain bacteria species. The authors found that in a 3D environment, the two different probiotics behaved differently at fighting the potentially pathogen species [66].

In a parallel work, Costello et al, employed the PLGA scaffolds to study cellular growth, differentiation and drug absorption functionality of the Ca2 and HT29-MTX co-culture model. It was observed that the co-culture of both epithelial cell types onto the PLGA scaffolds mimicked the native morphology and differentiation profile observed in native intestinal tissue, as confirmed by the expression and production of differentiation markers and by the secretion of mucus as well [67].

Other works gave origin to scaffolds for intestinal in vitro models not necessarily recreating the villi architecture, but also giving cues to the cells to behave similarly to the

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in vivo case.

A recent study by Dosh et al [68]. aimed at investigating the potential of three different hydrogel scaffolds to support the 3D culture of Caco-2 and HT29-MTX cells and evaluate their ability to stimulate villi formation. Here, alginate hydrogels were investigated, and cells were cultured in different set-ups as well as under static or dynamic conditions for up to 21 days. Caco-2 cell viability was increased when layered on the synthetic hydrogel scaffolds but reduced when suspended within the synthetic hydrogels, whereas HT29-MTX maintained similar viability in both conditions. Furthermore, cells cultured in and on alginate hydrogel scaffolds formed multilayer spheroid structures, while the cells layered on synthetic hydrogels formed villus-like structures [68].

With a different perspective and different scale approach, Chen et al. [69] established a 3D porous silk protein scaffolding system, containing a geometrically-engineered hollow lumen. The hollow channel of the 3D scaffolds was used to house Caco-2 and HT29-MTX cells, while the porous bulk space was used to culture primary human intestinal myofibroblasts (H-InMyoFibs) embedded in a collagen gel. This scaffold and respective cell culture set-up (illustrated in Figure 10) demonstrated to induce typical physiological responses by favoring continuous accumulation of mucous secretions on the epithelium, by establishing low oxygen tension in the lumen, and by allowing interaction with gut-colonizing bacteria. Moreover, this 3D intestinal model enabled months-long maintenance of the tissue function and cell phenotype [69].

Figure 10 - Production and cell arrangement of the scaffolds (patterned and not patterned) taken from Chen et al [69].

Continuing the work on the PLGA scaffolds previously mentioned in this section, Shaffiey et al. [70] investigated the growth and differentiation of intestinal cells on a novel tubular configuration scaffold. This time, the researchers used intestinal stem cells and tested the cell responses both in vitro and in vivo (with implantation in animal models). The results indicate that the differentiated into crypt-villi structures on the scaffold, and the scaffold coverage was enhanced by coculture with myofibroblasts, macrophages and probiotic bacteria. Remarkably, the implanted scaffolds enhanced mucosal regeneration

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3.5 Bioreactors for mechanical stimulation of cells

The cellular response to mechanical stimulation depends upon the type of force applied, for instance, tensile and compressive forces should be applied perpendicular to the surface of the cell or 3D construct; shear forces should be applied parallel to the cell or 3D construct surface; while strains can be applied by deforming an elastic cell substrate. The cellular response also depends on the magnitude, frequency and duration of the applied stimuli. Trying to understand the role of a certain physical force in cell and tissue behavior, researchers apply physiologically relevant mechanical stimuli at the cell and tissue level using engineered devices which have been designed to control the temporal, spatial and intensity of the force parameter [58]. The present section is dedicated to describing some examples of engineered systems for mechanical stimulation of cells.

Some techniques to study mechanotransduction phenomena in cells in their physiological microenvironment include the application of compression, hydrostatic pressure, tension and shear stress to cell monolayers, or tissue fragments. Since the 1970s mechanical devices have been used to analyze the role of static and dynamic compression in cell growth and metabolism [71]. The effect of hydrostatic pressure in cell growth and differentiation has been studied both in 2D and 3D conditions through the application of a transmembrane pressure plated on a porous substrate, or through directing compressed air or fluid over a culture of cells [72]. On the other hand, the response of cells to tensile stress has been relying on the application of static or cyclic, axial or biaxial strains to monolayers of cells cultured on deformable membranes or 3D scaffolds. Finally, shear stress has been applied to cell cultures through different flow chambers, which use either pressure-driven systems that apply a parabolic laminar flow profile, or flow systems that apply a uniform shear stress with a linear flow profile [73]. Additionally, and as a more recent approach, the response of individual cells to mechanic stimuli has been explored. This research line uses advanced devices that are able to apply pico- or nano-Newton forces to individual cell or even cellular elements, such it is the case of atomic force microscopy and traction force microscopy [74]. Nevertheless, these last techniques are not exactly suitable to study the dynamics of a complex tissue, as it is the scope of the of this thesis.

3.5.1. Flow for cell cultures

Medium flow is applied to the cell culture system by using bioreactors. In this context, bioreactors are in vitro culture systems that ensure cell survival through adequate delivery of essential nutrients throughout the tissue engineered construct; they also guide tissue structure and organization, and eventually function through the application of chemical and mechanical stimuli [75]. One important reason for their use is that oxygen, critical in all aerobic metabolic cycles, is often the limiting factor when culturing 3D cellular constructs in vitro (especially in the case of non-porous systems which rely on gradient-driven passive diffusion). The reason for this arises from the difficulty in bringing

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sufficient amounts of oxygen to the cells because of its poor solubility in culture media[19]. Indeed, several bioreactors were created to supply efficient oxygen and supply to 3D cell constructs that would not be supplied in an ‘old-fashioned’ culture manner due to mass and oxygen transfer limitations [76]. It was also reported in a study that the flow of a cell suspension would result on more efficiently and uniformly seeded scaffolds, compared with static seeding [77].

There are several types of bioreactors depending on their structure and purpose. Here, I will focus mainly on the effect of shear stress on different cell cultures, and finally, on intestinal models.

Various studies showed that different cells cultured in different bioreactors when exposed to shear stress modulate their behavior affecting tissue formation, being most of these studies about differentiation of stem cells (into bone, for instance), and expression changes in chondrocytes and endothelial cells. Different stem cell types have different optimal shear stress values [78], and by adjusting this parameter, different responses can be obtained. Regarding the employment of flow for intestinal in vitro models, several groups have been developing different systems and bioreactors that will be presented as follows.

3.5.1.1. Providing flow for in vitro intestinal models

Back in 1995, McBride et al. developed a bioreactor made of hollow fiber cassettes [79], that was later in 1999 adapted by the researcher to culture a cell line of human intestinal cells. The goal was to study the effect of chronic dietary or environmental toxin exposure. This set up allowed the researchers to perform a long-term study, in contrast with the acute toxic effect, back then usually verified in flask tissue culture [80]. Several years later, the hollow fiber bioreactor set-up is still applied to morphologically mimic the human small intestinal lumen. In 2013, Deng and his colleagues used porous hollow fibers of polyethersulfone (PES) to culture Caco-2 and study their differentiation and functioning. The set-up of the culture system is illustrated in Figure 11. The study revealed and accelerated expression of Caco-2 cell function on this system, showing the ability to simulate the original tissue microenvironment [81].

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Figure 11 – Representation of the hollow fiber bioreactor developed by Deng et al taken from [81].

To better simulate the conditions of digestion, including even a food matrix, a dynamic GIT model was conceived in 2005 by Mainville et al [82]. It consisted of two reactors, one simulating stomach conditions and the other simulating duodenum conditions, that would mimic the target organs, so that the authors could understand the interactions with the probiotics. The dynamic model was shown to better represent the events during upper GIT transit than the conventional methods [82]. Six years later, Tompkins et al. adapted this system to perform similar studies on the same subject and were able to conclude that some probiotics should be ingested at a specific time interval from the meal [83].

Continuing with the theme of the research on probiotic bacteria, a gastrointestinal tract simulator (GITS) bioreactor was conceived by Sumeri et al. The system consisted of a fermentation vessel equipped with diverse sensors and different pumps to provide the flow of diverse fluids. The work established that such GITS could be successfully used for evaluation of viability of probiotic bacteria and functionality of probiotic food [84]. Often, bioreactors are used to improve the culture conditions of primary cells, that are known for short survival times when cultured on the classic systems. In order to sustain in culture intestinal organoids harvested from rats, Kim et al. developed a perfusion bioreactor. The results confirmed the survival of intestinal epithelial cells seeded on the scaffolds and cultured in the bioreactor for two days [85]. A work by Pusch et al., consisted in using decellularized porcine jejunal segments co-cultured with Caco-2 cells and primary-isolated human microvascular endothelial cells (hMECs) in a dynamic bioreactor. Some of the data was comparable with classic Caco-2 testing results, while some results demonstrated that in the tissue-engineered segments cultured under dynamic conditions cells resembled normal primary enterocytes and showed increased permeability to tested substances, when compared with static cultures [86]. Recently, Schweinlin and his colleagues used previously established gut organoid technology, also with a natural ECM based on porcine small intestinal submucosa (SIS). The

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organoids, derived from intestinal crypts from healthy human small intestine, seeded on the scaffold differentiated into different intestinal cell types after 7 days . Additionally, the epithelial barrier function was tested under the effect of flow and with the co-culture of sub-epithelial fibroblasts as well. It was verified that the presence of the intestinal fibroblasts stabilized the barrier integrity and that the dynamic culture in a perfused bioreactor induced expression of differentiation markers on the epithelial cells, indicating that such complete system can sustain the culture of primary intestinal cells [87].

Still dealing with a perfusion system, but exploring microfluidics technology, in 2008 Kimura et al. developed a micro pumping system on-chip. Caco-2 cultured in the device for more than two weeks. Perfusion and transport measurements (using fluorescent compounds detected with an optical fiber system) were conducted, targeting the micro bioreactor for applications in toxicity testing and drug screening [88]. This system is represented in Figure 12.

Figure 12 – Representation of the microfluidics device developed by Kimura et al taken from [88]. A. Illustration of the different layers that constitute the device. B. Side-view representation of the final

device.

In 2010, microfluidics rather than emergent was an established technology, as shown by the works of Imura et al [89,90]. The authors developed a microchip-based system that mimicked the intestine. The microdevice was mainly composed of polydimethylsiloxane (PDMS) sheets, with microchannels fabricated using photolithography techniques, and the flow was regulated with a microsyringe pump. Caco-2 cells were cultured on the membrane in the microchip. The results of absorption tests run on cultured Caco-2 cells were consistent with those obtained using conventional methods, suggesting the suitability of the new system [89]. In the following year, the same research group used the developed microchip to integrate micromodels of tissues – a component for the intestine (using Caco-2 cells) and another one for the liver (using HepG2 cells). The authors tested the intestinal absorption, hepatic metabolism, and bioactivity of different substances and claimed the feasibility of the operations on their device, reducing time and cell consumption compared to the classic in vitro assays [90]. Some years after, with a

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similar approach, Bricks et al. joined cell culture inserts and microfluidic biochips in an integrated fluid platform to study the interaction between intestine and liver, using Caco-2 and HepGCaco-2, respectively. This work revealed that the integrity, viability and metabolism of both cell types were maintained and that the co-culture system allowed for biotransformation of a tested compound [91].

Of special interest for my research, the work of Giusti et al [92] assessed a novel two-chamber millifluidic bioreactor for the culture of intestinal epithelial cells. After analyzing the fluidic dynamics and pressure gradients for different combination of flow rates using, computational models, Caco-2 cells were cultured on the device until they fully differentiated. The authors verified that the dynamic conditions led to an increase in barrier integrity values and in expression of tight-junctions markers, in respect to the static controls. Interestingly, the permeability of the cell barrier to the tested compound was higher in dynamic conditions, suggesting that the bioreactor could be used to perform drug delivery and nanomaterial toxicity studies on different barrier tissues [92].

Bringing together the presence of dynamic conditions and the topography of the intestinal epithelium (in this case, crypts or villi), some authors developed sophisticated systems to recreate the microenvironment of the epithelium in study.

Wang et al. proposed a microengineered device to culture colonic crypts and colonoids (multi-cell spheroids isolated from the colon). A platform was fabricated from PDMS containing fluidic layers separated by an array of cylindrical micro-wells. Fluid and growth factors were supplied, and it was verified that colonoid growth, stem cell number and morphology of the cells on the array was like those under static culture conditions. Further results confirmed that the diverse differentiated cell-types of the colon formed on the array, showing the suitability of this "colon-on-a-chip" for reproducing organ-level function of the colon for controlled experiments [93].

Last year, Costello et al. developed in vitro artificial small intestines. In this work, a small intestinal bioreactor was constructed, using polymeric scaffolds that mimicked the 3D topography of the tissue, and provided with fluid flow. The obtained data indicated that the presence of flow induce closer to physiological condition values on the tightness of the cell barrier, comparing with static conditions. It was also verified an increase in cell proliferation and that some cell responses varied according to different regions of the construct and according to different tested flow rates as well [94].

3.5.2. Mechanical stretch for cell cultures

As any other mechanical force, stretch can be an important modulator of cell physiology. Different studies have shown that cyclic mechanical stretch induced proliferation, increased tissue organization, and enhanced mechanical properties on several cell types, as it is briefly illustrated in the following section.

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3.5.2.1. Providing mechanical stretching for in vitro intestines

When it comes to the intestine, the epithelium should be affected by the repetitive deformation during peristaltic distension and contraction and by the repetitive villus shortening. On a notable work by Basson et al, Caco-2 cells were cultured on a deformable membrane and subjected to 10% strains and it was verified that the cyclic strain stimulated proliferation. This response was higher in the membrane periphery where strain was maximal and, furthermore, it modulated the expression of specific brush border enzymes. The authors concluded that mechanically-induced strains at a physiological frequency and magnitude enhanced proliferation and modulated the differentiation of this cell line in an amplitude-dependent way [95]. Later, the molecular pathways that led to the reported effects were investigated and the proteins PKC and tyrosine kinase were pointed as regulators of intestinal epithelium proliferation and brush-border enzyme activity upon cyclic deformation [17].

Using a rat in vivo model, Safford and his colleagues suggested that mechanical tension induced intestinal growth. Their results indicated that the applied mechanical tension led to an increase in Paneth cells numbers, that caused proliferation and reorganization of the mucosa and muscularis propria. In addition, the increased intestinal length corresponded to an increase of enzymatic activity, suggesting a potential augmented absorption of the stretched bowels [96].

This time using an in silico model, more specifically a computational intestinal organoid culture model, Buske et al. studied the role of biomechanics on the stem cell niche formation in the gut. The researchers improved a previous computational model of the intestinal tissue [97] by adding a flexible basal membrane that assigned a bending modulus to the organoid surface. According to the test results, the proliferation induced shape changes leading to the formation of crypt-like domains. The spontaneous local tissue curvature could be a regulatory factor in stem cell organization [98], illustrating once more the effects of cell deformation on tissue differentiation.

Back to in vitro models of Caco-2 cells, a more recent study by Samak et al. explored the impact of cyclic stretch on tight junction and adherens junction integrity. The results indicated that due to activation of specific signaling pathways, those apical cell junctions were disrupted, as supported by the re-organization of the junction’s proteins and increased paracellular permeability [99].

Perhaps one of the currently more advanced in vitro models of the human intestine is the ‘human gut-on-a-chip’ by Kim et al. It consists of a microfluidic device that contemplates both the shear stress induced by fluid flow and the cell stretching induced by a deformable membrane. The device is composed of two microfluidic channels separated by a porous flexible membrane coated with ECM that was seeded with Caco-2 cells, as represented on Figure 13. The microenvironment of the intestinal epithelium was mimicked by using a medium flow at a rate of 30 μL h−1

that produced low shear stress (0.02 dyne cm−2), and by applying cyclic strain (10%; 0.15 Hz) that imitated physiological peristaltic motions. This system allowed for a quick polarization of the epithelium that spontaneously grown into folds

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Figure 13 - A. Representation of the gut-on-a-chip constitution. B. picture of the device, by Kim et al

[100].

Later, this same system was used to co-culture commensal microbes in contact with the intestinal epithelium cells for more than a week. The obtained data reveled that immune cells and endoxins together stimulated epithelial cells to produce proinflammatory cytokines that can induce villus injury and compromise intestinal barrier function. This showed that the chip can also be used to study interaction between microbiome and intestinal pathophysiology in a controlled environment [101].

This chapter reviewed different models able to recapitulate one or several features of the intestinal microenvironment. Each in vitro model poses its own advantages and limitations that reflect on their suitability for a certain application but not for some other. Accordingly, Annex A summarizes the current human cells-based intestinal in vitro models of the intestine with respective features and applications.

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Porous scaffolds that replicate the architecture of the

intestinal villi

Topography can elicit different cell responses such as cell orientation, rates of movement and cell activation, suggesting that by tuning topographical cues, one can control cell fate in an in vitro environment [102,103].

As presented before, the regulation of topographical cues can be accomplished using 3D structures known as cell scaffolds - one of the foundations of tissue engineering. In this context, scaffolds act as a template for tissue formation and are typically seeded with cells and occasionally growth factors, and/or subjected to biophysical stimuli using a bioreactor. Bioreactors are a device, tool or system that applies different types of mechanical or chemical stimuli to cells [104]. For any kind of application, a scaffold must be designed considering certain aspects: biocompatibility, biodegradability, necessary mechanical properties, architecture, and manufacturing technology [104]. Biocompatibility is the basic requirement since the chemical elements of the scaffold must induce molecular recognition from the cells so that they can adhere and proliferate; biodegradable polymers are a common choice for the composition of a cell scaffold. The mechanical properties and degradation kinetics should be adapted to the specific tissue engineering application to guarantee the required mechanical functions. Furthermore, pore distribution, the amount of exposed surface area, and porosity play an important role in the adequate development of cellular process [105]. Scaffolds can be made porous by distinct methods, as will be presented in this section. For the application proposed in this thesis, a porous scaffold could be of special interest to recreate the investigated topography and, at the same time, to provide a permeable interface that allows exchange of material between the apical and basal compartments

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Regarding the creation of 3D structures, nowadays there are different fabrication techniques that have been explored to develop sophisticated cell scaffolds. Generally, conventional fabrication techniques such as salt leaching, gas forming, phase separation, and freeze-drying (presented herein) do not enable precise control of internal scaffold architecture or the fabrication of complex architectures; additionally, the required use of toxic solvents may result in biocompatibility issues if they are not completely removed. [106] Alternatively, the production of microstructures with precision can be accomplished by methods that employ microengineered platforms.

On the basis of their working principle, such methods can be grouped in photomask-based methods, micromold based methods and rapid prototyping (RP) based methods. Rapid prototyping techniques use computer-aided design (CAD) modeling and have better design repeatability, consistency and control of scaffolds architecture at both micro and macro levels [106]. Photolabile approaches and high temperature approaches belong to this group of techniques. Both employ the same basic processes: first the construct is modeled using CAD, then the designed object is converted into a compatible format, and finally, the construct is fabricated layer-by layer by a 3D printing apparatus. In the photolabile RP approaches, as is the case of stereolitography (SLA), the layers of the 3D models are created from liquid photolabile polymers that can be cured when exposed to UV light. Differently, in high temperature approaches, each layer is created by deposition of the material across the plane of the layer cross section; then the material cools down and solidifies fixed to the previous layer. This method comprises two different tactics: one is by employing a powder of a suitable material and then applying high temperature to bond, such is the case of selective laser sintering (SLS) - where a laser is used to merge powdered materials in a solid structure; while the other method, called fused deposition modelling (FDM), uses a melted thermoplastic material which is then deposited by extrusion [107]. Nowadays there is also an emerging branch of tissue engineering that explores the possibility of printing cells in the construct, through different approaches that have to require conditions favorable for cell survival (high temperatures and toxic solvents are therefore excluded) [107]. Although rapid prototyping techniques may have several advantages, one of the main obstacles is that the number of biomaterials that can be processed by rapid prototyping is limited compared to conventional techniques [106].

Regarding traditional methods, although they cannot provide structures with precisely controlled architecture, some procedures have been successfully used to fabricate porous structures. Such is the case of gas forming, where gas is used to create pores. The process consists in application of high-pressure gas through polymers previously compressed at high temperatures and then after a suitable period, reducing the pressure back to atmospheric. Freeze-drying can be also used to create pores since the sublimation of frozen water directly into the gas phase creates empty spaces in the polymer mesh. Using the electrospinning technique, the polymeric jet of fluid is ejected into a collector where electrospun fibers are randomly deposited, forming a non-compact structure. Another commonly used approach is to mix a porogen or salt crystals into the polymer mix, and

Engineering tools for providing mechanical cues to intestinal in vitro models