Correction: Developing an advanced gut on chip model enabling the study of epithelial cell/fibroblast interactions.

Correction for 'Developing an advanced gut on chip model enabling the study of epithelial cell/fibroblast interactions' by Marine Verhulsel et al., Lab Chip, 2021, 21, 365-377, https://doi.org/10.1039/d0lc00672f.

Precise and fast control of the dissolved oxygen level for tumor-on-chip.

In vitro cell cultures are most often performed in unphysiological hyperoxia since the oxygen partial pressure of conventional incubators is set at 141 mmHg (18.6%, close to ambient air oxygen 20.1%). This value is higher than human tissue oxygen levels, as the in vivo oxygen partial pressures range from 104 mmHg (lung alveoli) to 8 mmHg (skin epidermis). Importantly, under pathological conditions such as cancer, cells can experience oxygen pressure lower than the healthy tissue. Although hypoxic incubators can regulate gas oxygen, they do not take into account the dissolved oxygen concentration in the cell culture medium. In the context of organ on chip and micro-physiological system development, we present here a new system, called Oxalis (OXygen ALImentation System) that allows fine control of the dissolved oxygen level in the cell culture medium. Oxalis regulates simultaneously the gas composition and the inlet reservoir pressure by modulating the pneumatic valve opening. This dual regulation allows both the pressure driven liquid flowrate and the level of oxygen dissolved in the chip to be controlled independently. Oxalis offers unprecedented features such as an oxygen equilibration time lower than 3 minutes and an accuracy of 3 mmHg. These performances can be reached for chip perfusion flow as low as 1 µL min-1. This low flow rate allows the shear stress experienced by the cells in the chip to be accurately controlled. In addition, the system enables modulation of the pH in the cell culture medium through the modulation of CO2. The fine control and monitoring of both O2 and pH pave the way for new precise investigations on physiological and pathological biological processes. Using Oxalis in the context of tumor-on-chip, we demonstrate the capacity of the system to recapitulate hypoxia-induced gene expression, offering an innovative strategy for future studies on the role of hypoxia in malignant progression and drug resistance.

Developing an advanced gut on chip model enabling the study of epithelial cell/fibroblast interactions.

Organoids are widely used as a model system to study gut pathophysiology; however, they fail to fully reproduce the complex, multi-component structure of the intestinal wall. We present here a new gut on chip model that allows the co-culture of primary epithelial and stromal cells. The device has the topography and dimensions of the mouse gut and is based on a 3D collagen I scaffold. The scaffold is coated with a thin layer of laminin to mimic the basement membrane. To maintain the scaffold structure while preserving its cytocompatibility, the collagen scaffold was rigidified by threose-based post-polymerization treatment. This treatment being cytocompatible enabled the incorporation of primary intestinal fibroblasts inside the scaffold, reproducing the gut stromal compartment. We observed that mouse organoids, when deposited into crypts, opened up and epithelialized the scaffold, generating a polarized epithelial monolayer. Proper segregation of dividing and differentiated cells along the crypt-villus axis was achieved under these conditions. Finally, we show that the application of fluid shear stress allows the long-term culture of this intestinal epithelium. Our device represents a new biomimetic tool that captures key features of the gut complexity and could be used to study gut pathophysiology.

Sliding walls: a new paradigm for fluidic actuation and protocol implementation in microfluidics.

Currently, fluidic control in microdevices is mainly achieved either by external pumps and valves, which are expensive and bulky, or by valves integrated in the chip. Numerous types of internal valves or actuation methods have been proposed, but they generally impose difficult compromises between performance and fabrication complexity. We propose here a new paradigm for actuation in microfluidic devices based on rigid or semi-rigid walls with transversal dimensions of hundreds of micrometres that are able to slide within a microfluidic chip and to intersect microchannels with hand-driven or translation stage-based actuation. With this new concept for reconfigurable microfluidics, the implementation of a wide range of functionalities was facilitated and allowed for no or limited dead volume, low cost and low footprint. We demonstrate here several fluidic operations, including on/off or switch valving, where channels are blocked or reconfigured depending on the sliding wall geometry. The valves sustain pressures up to 30 kPa. Pumping and reversible compartmentalisation of large microfluidic chambers were also demonstrated. This last possibility was applied to a "4D" migration assay of dendritic cells in a collagen gel. Finally, sliding walls containing a hydrogel-based membrane were developed and used to concentrate, purify and transport biomolecules from one channel to another, such functionality involving complex fluidic transport patterns not possible in earlier microfluidic devices. Overall, this toolbox is compatible with "soft lithography" technology, allowing easy implementation within usual fabrication workflows for polydimethylsiloxane chips. This new technology opens the route to a variety of microfluidic applications, with a focus on simple, hand-driven devices for point-of-care or biological laboratories with low or limited equipment and resources.

A new biomimetic assay reveals the temporal role of matrix stiffening in cancer cell invasion.

Tumor initiation and growth is associated with significant changes in the surrounding tissue. During carcinoma progression, a global stiffening of the extracellular matrix is observed and is interpreted as a signature of aggressive invasive tumors. However, it is still unknown whether this increase in matrix rigidity promotes invasion and whether this effect is constant along the course of invasion. Here we have developed a biomimetic in vitro assay that enabled us to address the question of the importance of tissue rigidity in the chronology of tumor invasion. Using low concentrations of the sugar threose, we can effectively stiffen reconstituted collagen I matrices and control the stiffening in time with no direct effect on residing cells. Our findings demonstrate that, depending on the timing of its stiffening, the extracellular matrix could either inhibit or promote cancer cell invasion and subsequent metastasis: while matrix stiffening after the onset of invasion promotes cancer cell migration and tumor spreading, stiff matrices encapsulate the tumor at an early stage and prevent cancer cell invasion. Our study suggests that adding a temporal dimension in in vitro models to analyze biological processes in four dimensions is necessary to fully capture their complexity.

Transient microfluidic compartmentalization using actionable microfilaments for biochemical assays, cell culture and organs-on-chip.

We report here a simple yet robust transient compartmentalization system for microfluidic platforms. Cylindrical microfilaments made of commercially available fishing lines are embedded in a microfluidic chamber and employed as removable walls, dividing the chamber into several compartments. These partitions allow tight sealing for hours, and can be removed at any time by longitudinal sliding with minimal hydrodynamic perturbation. This allows the easy implementation of various functions, previously impossible or requiring more complex instrumentation. In this study, we demonstrate the applications of our strategy, firstly to trigger chemical diffusion, then to make surface co-coating or cell co-culture on a two-dimensional substrate, and finally to form multiple cell-laden hydrogel compartments for three-dimensional cell co-culture in a microfluidic device. This technology provides easy and low-cost solutions, without the use of pneumatic valves or external equipment, for constructing well-controlled microenvironments for biochemical and cellular assays.

A review of microfabrication and hydrogel engineering for micro-organs on chips.

This review highlights recent trends towards the development of in vitro multicellular systems with definite architectures, or "organs on chips". First, the chemical composition and mechanical properties of the scaffold have to be consistent with the anatomical environment in vivo. In this perspective, the flourishing interest in hydrogels as cellular substrates has highlighted the main parameters directing cell differentiation that need to be recapitulated in artificial matrix. Another scaffold requirement is to act as a template to guide tissue morphogenesis. Therefore specific microfabrication techniques are required to spatially pattern the environment at microscale. 2D patterning is particularly efficient for organizing planar polarized cell types such as endothelial cells or neurons. However, most organs are characterized by specific sub units organized in three dimensions at the cellular level. The reproduction of such 3D patterns in vitro is necessary for cells to fully differentiate, assemble and coordinate to form a coherent micro-tissue. These physiological microstructures are often integrated in microfluidic devices whose controlled environments provide the cell culture with more life-like conditions than traditional cell culture methods. Such systems have a wide range of applications, for fundamental research, as tools to accelerate drug development and testing, and finally, for regenerative medicine.