Engineering Complex Intestinal Epithelial Tissue Models

Periodic Reporting for period 4 - COMIET (Engineering Complex Intestinal Epithelial Tissue Models)

Période du rapport: 2020-06-01 au 2021-05-31

1. Almost all our fundamental knowledge about cell biology comes from in vitro assays performed on cell growing in plastic wells, which leads to results that are not always useful to translate to the in vivo scenario. For that reason, there is a growing trend to shift from the simplistic approaches of “cells on a petri dish” to mini-organs for in vitro studies. COMIET aims to accelerate this trend by providing a new model of gut tissue to advance the in vitro modelling of diseases, the preclinical screening for drug efficacy, absorbance and toxicity and the understanding of organ development.

2. Today, advancing a drug candidate to regulatory approval takes roughly 12 to 15 years and costs an estimated $800 to $900 million on average. As over 50% of drug candidates fail early, discarding of compounds in the in vitro preclinical tests saves costs, time and reduces the ethical concerns of indiscriminate usage of animal testing. On the other hand, the benefits of having an advanced model of gut to mimic digestive diseases and disorders is huge. The quality of life of people affected with inflammatory bowel disease is significantly lower than in healthy persons. Since therapeutic approaches are only symptomatic, the development of in vitro human intestinal models might contribute to shed light on the basic biology of these diseases by proposing new therapeutic strategies.

3. In this context, COMIET has made use of microengineering technologies and adult stem cells to develop artificial intestinal tissues that mimic the physiological characteristics found in vivo to open new areas of research on human intestinal diseases. The novel in vitro models of intestinal epithelial tissues aim to address intestinal biology such as cell renewal and migration, multicell-type differentiation, and cell-cell interactions to go beyond the state-of-the-art 3D minigut (organoid) in vitro models. The cell culture platforms proposed aimed to provide physiologically relevant and highly reproducible data, being compatible with conventional cell culture assays and high-throughput testing. To achieve this, we have developed an experimental setup that combines microfabrication technologies, tissue engineering and advances in intestinal stem cell research, exploiting stem cell self-organizing characteristics.

1. We have successfully implemented a simple and cost-effective method to fabricate a model of gut tissue with physiological-resembling anatomic architecture and dimensions that can be easily integrated into standard cell culture platforms. Two microfabrication techniques have been implemented: dynamic photolithography (Castaño et al., Biofabrication 2019; Altay et al., Front. Bioeng. Biotechnol. 2020; G. Altay, PhD thesis 2019) and digital light processing (DLP). This last technique is the basis of an ERC-PoC (GUT3DPLATE) which is currently running to explore the benefits of these substrates into market niches. This has delayed the publication of the results (Torras et al., to be submitted) to guarantee IP protection properly. Our progress in microfabrication of hydrogel materials has also been recognized by being invited to submit a spotlight review paper in ACS Applied Mater. & Interfaces (Vera et al., 2021) and leading a topic in Frontiers in Bioeng. & Biotechnol. (ebook). In addition, I have been invited to present this work in multiple international conferences (ESOF 2018; CRS Annual Meeting 2018 (Award to the best postdoc of the oral delivery group); XXVII International Materials Research Congress - IMRC 2018 (invited presentation); NanoBioConference 2018; NICE Conference 2018, MRS Fall meeting 2020, Biomaterials World Conference 2020 or microTAS 2021, among others).
2. We have demonstrated that intestinal cells cultured on our substrates behave in a more physiological manner than cells grown on conventional substrates. They better recreate the intestinal barrier properties: adherence and invasion capacity of E. coli LF82 pathogenic bacterial (García-Diaz et al., CRS Conference 2019), permeability and drug absorption properties (Castaño et al., Biofabrication 2019) and allow mimicking the interaction between different cell types of the intestinal mucosa (Vila et al., Biofabrication 2020), which is crucial to disease modelling (A. Vila, PhD thesis 2020). To better exploit this result we have established an on-going collaboration with a local hospital (Hospital Vall d’Hebron).
3. We have successfully developed and characterized several hydrogel formulations that can be used as bioinks in light-based applications (Castaño et al., Biofabrication 2019; Vila et al., Biofabrication 2020; Borgheti et al., in preparation). Aside of the scientific dissemination, we have established an agreement with a Spanish company that develops bioprinting instruments (Regemat 3D) to transfer a bioink specific formulation to their product catalogue.
4. Organoid-derived intestinal epithelial cells can grow on our 3D hydrogels and, upon delivering specific molecular gradients through a microfluidic device, cells are compartmentalized thus recreating their position in the in vivo tissue (G. Altay, PhD thesis 2019; Altay et al., 10.1101/2021.12.13.472418; Martinez et al, Biomaterials Conf. 2020; Martinez et al., MRS fall conf. 2020). We have established a protocol to fabricate the scaffold, the device and to “open-up” intestinal organoids onto monolayers (Altay et al. Scie Reports 2019; Altay et al., Bio-protocol 2020).
5. We successfully developed 2D approaches that have proven to be useful technological tools for the study of relevant factors recognized to affect intestinal cell behavior such as the stiffness of the substrate (Comelles et al., Biofabrication 2020), the patterning of relevant biomolecules identified in the intestinal development and homeostasis (Hortigüela et al., Nano Letters 2018; Cutrale et al., Nat. Protocols 2019; E. Larrañaga, PhD thesis 2021) and new 3D set-ups to study epithelial-stromal interactions (Fernández-Majada et al., 10.1101/2021.05.28.446131).

In COMIET, we have provided an alternative cell culture system that shows as advantages to the standard state of the art: (i) reproduces the 3D architecture of the tissue structure, (ii) it is as soft as the intestinal tissue, (iii) the fabrication process is easy and cost effective and can be adapted to standard cell culture platforms, (iv) provides barrier resistance values closer to those reported in vivo. On the other hand, the impact of digestive diseases and disorders is huge and have enormous societal and economic costs, so the development of in vitro human intestinal models might contribute to shed light on the basic biology of these diseases and contribute to propose new therapeutic strategies. COMIET has contributed to this field by providing a new protocol to produce intestinal epithelial monolayers from organoids and unique technological set-ups to study in a systematic manner the cellular, physical and biomolecular factors driving cell behaviour. These new systems might be used in basic biology applications, drug absorption tests and disease modelling. It has also opened new possibilities in regenerating intestinal tissues or personalizing cancer healthcare.

Intestinal organoids-derived single cells grown on microstructure hydrogels under biochemical spatia

Caco-2 cells form a mature epithelial monolayer on the 3D microstructured scaffolds

Periodic Reporting for period 4 - COMIET (Engineering Complex Intestinal Epithelial Tissue Models)

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