Collagen I Based Enzymatically Degradable Membranes for Organ-on-a-Chip Barrier Models.

Organs-on-chips are microphysiological in vitro models of human organs and tissues that rely on culturing cells in a well-controlled microenvironment that has been engineered to include key physical and biochemical parameters. Some systems contain a single perfused microfluidic channel or a patterned hydrogel, whereas more complex devices typically employ two or more microchannels that are separated by a porous membrane, simulating the tissue interface found in many organ subunits. The membranes are typically made of synthetic and biologically inert materials that are then coated with extracellular matrix (ECM) molecules to enhance cell attachment. However, the majority of the material remains foreign and fails to recapitulate the native microenvironment of the barrier tissue. Here, we study microfluidic devices that integrate a vitrified membrane made of collagen-I hydrogel (VC). The biocompatibility of this membrane was confirmed by growing a healthy population of stem cell derived endothelial cells (iPSC-EC) and immortalized retinal pigment epithelium (ARPE-19) on it and assessing morphology by fluorescence microscopy. Moreover, VC membranes were subjected to biochemical degradation using collagenase II. The effects of this biochemical degradation were characterized by the permeability changes to fluorescein. Topographical changes on the VC membrane after enzymatic degradation were also analyzed using scanning electron microscopy. Altogether, we present a dynamically bioresponsive membrane integrated in an organ-on-chip device with which disease-related ECM remodeling can be studied.

Microfluidic organ-on-a-chip model of the outer blood-retinal barrier with clinically relevant read-outs for tissue permeability and vascular structure.

The outer blood-retinal barrier (oBRB) tightly controls the transport processes between the neural tissue of the retina and the underlying blood vessel network. The barrier is formed by the retinal pigment epithelium (RPE), its basal membrane and the underlying choroidal capillary bed. Realistic three-dimensional cell culture based models of the oBRB are needed to study mechanisms and potential treatments of visual disorders such as age-related macular degeneration that result from dysfunction of the barrier tissue. Ideally, such models should also include clinically relevant read-outs to enable translation of experimental findings in the context of pathophysiology. Here, we report a microfluidic organ-on-a-chip model of the oBRB that contains a monolayer of human immortalized RPE and a microvessel of human endothelial cells, separated by a semi-permeable membrane. Confluent monolayers of both cell types were confirmed by fluorescence microscopy. The three-dimensional vascular structures within the chip were imaged by optical coherence tomography: a medical imaging technique, which is routinely applied in ophthalmology. Differences in diameters and vessel density could be readily detected. Upon inducing oxidative stress by treating with hydrogen peroxide (H2O2), a dose dependent increase in barrier permeability was observed by using a dynamic assay for fluorescence tracing, analogous to the clinically used fluorescence angiography. This organ-on-a-chip of the oBRB will allow future studies of complex disease mechanisms and treatments for visual disorders using clinically relevant endpoints in vitro.

Tunable Microstructured Membranes in Organs-on-Chips to Monitor Transendothelial Hydraulic Resistance.

Tissue engineering is an interdisciplinary field, wherein scientists from different backgrounds collaborate to address the challenge of replacing damaged tissues and organs through the in vitro fabrication of functional and transplantable biological structures. Because the development and optimization of tissue engineering strategies rely on the complex interaction of cells, materials, and the physical-chemical tissue microenvironment, there is a need for experimental models that allow controlled studies of these aspects. Organs-on-chips (OOCs) have recently emerged as in vitro models that capture the complexity of human tissues in a controlled manner, while including functional readouts related to human organ physiology. OOCs consist of multiple microfluidic cell culture compartments, which are interfaced by porous membranes or hydrogels in which human cells can be cultured, thereby providing a controlled culture environment that resembles the microenvironment of a certain organ, including mechanical, biochemical, and geometrical aspects. Because OOCs provide both a well-controlled microenvironment and functional readouts, they provide a unique opportunity to incorporate, evaluate, and optimize materials for tissue engineering. In this study, we introduce a polymeric blend membrane with a three-dimensional double-porous morphology prepared from a poly(ɛ-caprolactone)-chitosan blends (PCL-CHT) by a modified liquid-induced phase inversion technique. The membranes have different physicochemical, microstructural, and morphological properties depending on different PCL-CHT ratios. Big surface pores (macrovoids) provide a suitable microenvironment for the incorporation of cells or growth factors, whereas an interconnected small porous (macroporous) network allows transfer of essential nutrients, diffusion of oxygen, and removal of waste. Human umbilical vein endothelial cells were seeded on the blend membranes embedded inside an OOC device. The cellular hydraulic resistance was evaluated by perfusing culture medium at a realistic transendothelial pressure of 20 cmH2O or 2 kPa at 37°C after 1 and 3 days postseeding. By introducing and increasing CHT weight percentage, the resistance of the cellular barrier after 3 days was significantly improved. The high tuneability over the membrane physicochemical and architectural characteristics might potentially allow studies of cell-matrix interaction, cell transportation, and barrier function for optimization of vascular scaffolds using OOCs. Impact Statement Organs-on-chips (OOCs) offer interesting potential for progress in the treatment of diseases and injury in the growing field of tissue engineering and regenerative medicine. The article presents a new way to develop polymer membrane with a tunable microstructured morphology and to implement this biomaterial inside an OOC device. The reader should find measurements of the transendothelial hydraulic resistance in real time during endothelial cells culture: a simple and controlled way of mimicking human physiological condition for vascular tissue regeneration. This combination of novel biomaterial inside an OOC will explore innovative ideas in tissue engineering field.

Barriers-on-chips: Measurement of barrier function of tissues in organs-on-chips.

Disruption of tissue barriers formed by cells is an integral part of the pathophysiology of many diseases. Therefore, a thorough understanding of tissue barrier function is essential when studying the causes and mechanisms of disease as well as when developing novel treatments. In vitro methods play an integral role in understanding tissue barrier function, and several techniques have been developed in order to evaluate barrier integrity of cultured cell layers, from microscopy imaging of cell-cell adhesion proteins to measuring ionic currents, to flux of water or transport of molecules across cellular barriers. Unfortunately, many of the current in vitro methods suffer from not fully recapitulating the microenvironment of tissues and organs. Recently, organ-on-chip devices have emerged to overcome this challenge. Organs-on-chips are microfluidic cell culture devices with continuously perfused microchannels inhabited by living cells. Freedom of changing the design of device architecture offers the opportunity of recapitulating the in vivo physiological environment while measuring barrier function. Assessment of barriers in organs-on-chips can be challenging as they may require dedicated setups and have smaller volumes that are more sensitive to environmental conditions. But they do provide the option of continuous, non-invasive sensing of barrier quality, which enables better investigation of important aspects of pathophysiology, biological processes, and development of therapies that target barrier tissues. Here, we discuss several techniques to assess barrier function of tissues in organs-on-chips, highlighting advantages and technical challenges.