Scaled-up Microfluidic Lung Assist Device for Artificial Placenta Application with High Gas Exchange Capacity.

Premature neonates with underdeveloped lungs experience respiratory issues and need respiratory support, such as mechanical ventilation or extracorporeal membrane oxygenation (ECMO). The "artificial placenta" (AP) is a noninvasive approach that supports their lungs and reduces respiratory distress, using a pumpless oxygenator connected to the systemic circulation, and can address some of the morbidity issues associated with ECMO. Over the past decade, microfluidic blood oxygenators have garnered significant interest for their ability to mimic physiological conditions and incorporate innovative biomimetic designs. Achieving sufficient gas transfer at a low enough pressure drop for a pumpless operation without requiring a large volume of blood to prime such an oxygenator has been the main challenge with microfluidic lung assist devices (LAD). In this study, we improved the gas exchange capacity of our microfluidic-based artificial placenta-type LAD while reducing its priming volume by using a modified fabrication process that can accommodate large-area thin film microfluidic blood oxygenator (MBO) fabrication with a very high gas exchange surface. Additionally, we demonstrate the effectiveness of a LAD assembled by using these scaled-up MBOs. The LAD based on our artificial placenta concept effectively increases oxygen saturation levels by 30% at a flow rate of 40 mL/min and a pressure drop of 23 mmHg in room air, which is sufficient to support partial oxygenation for 1 kg preterm neonates in respiratory distress. When the gas ambient environment was changed to pure oxygen at atmospheric pressure, the LAD would be able to support premature neonates weighing up to 2 kg. Furthermore, our experiments reveal that the LAD can handle high blood flow rates of up to 150 mL/min and increase oxygen saturation levels by ∼20%, which is equal to an oxygen transfer of 7.48 mL/min in an enriched oxygen environment and among the highest for microfluidic AP type devices. Such performance makes this LAD suitable for providing essential support to 1-2 kg neonates in respiratory distress.

A roadmap for developing and engineering in vitro pulmonary fibrosis models.

Idiopathic pulmonary fibrosis (IPF) is a severe form of pulmonary fibrosis. IPF is a fatal disease with no cure and is challenging to diagnose. Unfortunately, due to the elusive etiology of IPF and a late diagnosis, there are no cures for IPF. Two FDA-approved drugs for IPF, nintedanib and pirfenidone, slow the progression of the disease, yet fail to cure or reverse it. Furthermore, most animal models have been unable to completely recapitulate the physiology of human IPF, resulting in the failure of many drug candidates in preclinical studies. In the last few decades, the development of new IPF drugs focused on changes at the cellular level, as it was believed that the cells were the main players in IPF development and progression. However, recent studies have shed light on the critical role of the extracellular matrix (ECM) in IPF development, where the ECM communicates with cells and initiates a positive feedback loop to promote fibrotic processes. Stemming from this shift in the understanding of fibrosis, there is a need to develop in vitro model systems that mimic the human lung microenvironment to better understand how biochemical and biomechanical cues drive fibrotic processes in IPF. However, current in vitro cell culture platforms, which may include substrates with different stiffness or natural hydrogels, have shortcomings in recapitulating the complexity of fibrosis. This review aims to draw a roadmap for developing advanced in vitro pulmonary fibrosis models, which can be leveraged to understand better different mechanisms involved in IPF and develop drug candidates with improved efficacy. We begin with a brief overview defining pulmonary fibrosis and highlight the importance of ECM components in the disease progression. We focus on fibroblasts and myofibroblasts in the context of ECM biology and fibrotic processes, as most conventional advanced in vitro models of pulmonary fibrosis use these cell types. We transition to discussing the parameters of the 3D microenvironment that are relevant in pulmonary fibrosis progression. Finally, the review ends by summarizing the state of the art in the field and future directions.

Adhesive-Based Fabrication Technique for Culture of Lung Airway Epithelial Cells with Applications in Cell Patterning and Microfluidics.

This work describes a versatile and cost-effective cell culture method for micropatterning and growing adherent cells on porous membranes using pressure-sensitive double-sided adhesives. This technique also allows cell culture using conventional methods and their easy integration into microfluidic chip devices. Adhesives can be used to form different patterns of cultured cells, which can be used for cell proliferation and wound-healing models. To demonstrate the viability of our system, we evaluate the toxicity effect of five different adhesives on two distinct airway epithelial cell lines and show functional applications for cell patterning and microfluidic cell culture chip fabrication. We developed a sandwiched microfluidic device that enabled us to culture cells in a submerged condition and transformed it into a dynamic platform when required. The viability of cells and their inflammatory responses to IL-1ß stimulation were investigated. Our technique is applicable for conventional culturing of cells, widely available in biomedical research labs, while enabling the introduction of perfusion for an advanced dynamic cell culture model when needed.

A Robust Protocol for Decellularized Human Lung Bioink Generation Amenable to 2D and 3D Lung Cell Culture.

Decellularization efforts must balance the preservation of the extracellular matrix (ECM) components while eliminating the nucleic acid and cellular components. Following effective removal of nucleic acid and cell components, decellularized ECM (dECM) can be solubilized in an acidic environment with the assistance of various enzymes to develop biological scaffolds in different forms, such as sheets, tubular constructs, or three-dimensional (3D) hydrogels. Each organ or tissue that undergoes decellularization requires a distinct and optimized protocol to ensure that nucleic acids are removed, and the ECM components are preserved. The objective of this study was to optimize the decellularization process for dECM isolation from human lung tissues for downstream 2D and 3D cell culture systems. Following protocol optimization and dECM isolation, we performed experiments with a wide range of dECM concentrations to form human lung dECM hydrogels that were physically stable and biologically responsive. The dECM based-hydrogels supported the growth and proliferation of primary human lung fibroblast cells in 3D cultures. The dECM is also amenable to the coating of polyester membranes in Transwell™ Inserts to improve the cell adhesion, proliferation, and barrier function of primary human bronchial epithelial cells in 2D. In conclusion, we present a robust protocol for human lung decellularization, generation of dECM substrate material, and creation of hydrogels that support primary lung cell viability in 2D and 3D culture systems.

Surface Modification of PDMS-Based Microfluidic Devices with Collagen Using Polydopamine as a Spacer to Enhance Primary Human Bronchial Epithelial Cell Adhesion.

Polydimethylsiloxane (PDMS) is a silicone-based synthetic material used in various biomedical applications due to its properties, including transparency, flexibility, permeability to gases, and ease of use. Though PDMS facilitates and assists the fabrication of complicated geometries at micro- and nano-scales, it does not optimally interact with cells for adherence and proliferation. Various strategies have been proposed to render PDMS to enhance cell attachment. The majority of these surface modification techniques have been offered for a static cell culture system. However, dynamic cell culture systems such as organ-on-a-chip devices are demanding platforms that recapitulate a living tissue microenvironment's complexity. In organ-on-a-chip platforms, PDMS surfaces are usually coated by extracellular matrix (ECM) proteins, which occur as a result of a physical and weak bonding between PDMS and ECM proteins, and this binding can be degraded when it is exposed to shear stresses. This work reports static and dynamic coating methods to covalently bind collagen within a PDMS-based microfluidic device using polydopamine (PDA). These coating methods were evaluated using water contact angle measurement and atomic force microscopy (AFM) to optimize coating conditions. The biocompatibility of collagen-coated PDMS devices was assessed by culturing primary human bronchial epithelial cells (HBECs) in microfluidic devices. It was shown that both PDA coating methods could be used to bind collagen, thereby improving cell adhesion (approximately three times higher) without showing any discernible difference in cell attachment between these two methods. These results suggested that such a surface modification can help coat extracellular matrix protein onto PDMS-based microfluidic devices.

PHAIR: a biosensor for pH measurement in air-liquid interface cell culture.

In many biological systems, pH can be used as a parameter to understand and study cell dynamics. However, measuring pH in live cell culture is limited by the sensor ion specificity, proximity to the cell surface, and scalability. Commercially available pH sensors are difficult to integrate into a small-scale cell culture system due to their size and are not cost-effective for disposable use. We made PHAIR-a new pH sensor that uses a micro-wire format to measure pH in vitro human airway cell culture. Tungsten micro-wires were used as the working electrodes, and silver micro-wires with a silver/silver chloride coating were used as a pseudo reference electrode. pH sensitivity, in a wide and narrow range, and stability of these sensors were tested in common standard buffer solutions as well as in culture media of human airway epithelial cells grown at the air-liquid interface in a 24 well cell culture plate. When measuring the pH of cells grown under basal and challenge conditions using PHAIR, cell viability and cytokine responses were not affected. Our results confirm that micro-wire-based sensors have the capacity for miniaturization and detection of diverse ions while maintaining sensitivity. This suggests the broad application of PHAIR in various biological experimental settings.

A Pumpless Microfluidic Neonatal Lung Assist Device for Support of Preterm Neonates in Respiratory Distress.

Premature neonates suffer from respiratory morbidity as their lungs are immature, and current supportive treatment such as mechanical ventilation or extracorporeal membrane oxygenation causes iatrogenic injuries. A non-invasive and biomimetic concept known as the "artificial placenta" (AP) would be beneficial to overcome complications associated with the current respiratory support of preterm infants. Here, a pumpless oxygenator connected to the systemic circulation supports the lung function to relieve respiratory distress. In this paper, the first successful operation of a microfluidic, artificial placenta type neonatal lung assist device (LAD) on a newborn piglet model, which is the closest representation of preterm human infants, is demonstrated. This LAD has high oxygenation capability in both pure oxygen and room air as the sweep gas. The respiratory distress that the newborn piglet is put under during experimentation, repeatedly and over a significant duration of time, is able to be relieved. These findings indicate that this LAD has a potential application as a biomimetic artificial placenta to support the respiratory needs of preterm neonates.

An ultra-thin, all PDMS-based microfluidic lung assist device with high oxygenation capacity.

Preterm neonates with immature lungs require a lung assist device (LAD) to maintain oxygen saturation at normal levels. Over the last decade, microfluidic blood oxygenators have attracted considerable interest due to their ability to incorporate unique biomimetic design and to oxygenate in a physiologically relevant manner. Polydimethylsiloxane (PDMS) has become the main material choice for these kinds of devices due to its high gas permeability. However, fabrication of large area ultrathin microfluidic devices that can oxygenate sufficient blood volumes at clinically relevant flow rates, entirely made of PDMS, have been difficult to achieve primarily due to failure associated with stiction of thin PDMS membranes to each other at undesired locations during assembly. Here, we demonstrate the use of a modified fabrication process to produce large area ultrathin oxygenators entirely made of PDMS and robust enough to withstand the hydraulic conditions that are encountered physiologically. We also demonstrate that a LAD assembled from these ultrathin double-sided microfluidic blood oxygenators can increase the oxygen saturation level by 30% at a flow rate of 30 ml/min and a pressure drop of 21 mm Hg in room air which is adequate for 1 kg preterm neonates. In addition, we demonstrated that our LAD could withstand high blood flow rate of 150 ml/min and increase oxygen saturation by 26.7% in enriched oxygen environment which is the highest gas exchange reported so far by any microfluidic-based blood oxygenators. Such performance makes this LAD suitable to provide support to 1 kg neonate suffering from respiratory distress syndrome.

An ultra-thin highly flexible microfluidic device for blood oxygenation.

Many neonates who are born premature suffer from respiratory distress syndrome (RDS) for which mechanical ventilation and an extracorporeal membrane oxygenation (ECMO) device are used in treatment. However, the use of these invasive techniques results in higher risk of complications like bronchopulmonary dysplasia or requires surgery to gain vascular access. An alternative biomimetic approach is to use the umbilical cord as a vascular access and to connect a passive device to the baby that functions like a placenta. This concept, known as the artificial placenta, provides enough oxygenation and causes minimal distress or complications. Herein, we have developed a new artificial placenta-type microfluidic blood oxygenator (APMBO) with high gas exchange, low priming volume and low hydraulic resistance such that it can be operated only by pressure differential provided by the baby's heart. Mimicking the placenta, we have made our new device ultra-thin and flexible so that it can be folded into a desired shape without losing its capability for gas exchange and achieve a compact form factor. The ability to fold allowed optimization of connectors and reduced the overall priming volume to the sub-milliliter range while achieving a high oxygen uptake which would be sufficient for preterm neonates with a birth-weight of around 0.5 kg.

Steel reinforced composite silicone membranes and its integration to microfluidic oxygenators for high performance gas exchange.

Respiratory distress syndrome (RDS) is one of the main causes of fatality in newborn infants, particularly in neonates with low birth-weight. Commercial extracorporeal oxygenators have been used for low-birth-weight neonates in neonatal intensive care units. However, these oxygenators require high blood volumes to prime. In the last decade, microfluidics oxygenators using enriched oxygen have been developed for this purpose. Some of these oxygenators use thin polydimethylsiloxane (PDMS) membranes to facilitate gas exchange between the blood flowing in the microchannels and the ambient air outside. However, PDMS is elastic and the thin membranes exhibit significant deformation and delamination under pressure which alters the architecture of the devices causing poor oxygenation or device failure. Therefore, an alternate membrane with high stability, low deformation under pressure, and high gas exchange was desired. In this paper, we present a novel composite membrane consisting of an ultra-thin stainless-steel mesh embedded in PDMS, designed specifically for a microfluidic single oxygenator unit (SOU). In comparison to homogeneous PDMS membranes, this composite membrane demonstrated high stability, low deformation under pressure, and high gas exchange. In addition, a new design for oxygenator with sloping profile and tapered inlet configuration has been introduced to achieve the same gas exchange at lower pressure drops. SOUs were tested by bovine blood to evaluate gas exchange properties. Among all tested SOUs, the flat design SOU with composite membrane has the highest oxygen exchange of 40.32 ml/min m2. The superior performance of the new device with composite membrane was demonstrated by constructing a lung assist device (LAD) with a low priming volume of 10 ml. The LAD was achieved by the oxygen uptake of 0.48-0.90 ml/min and the CO2 release of 1.05-2.27 ml/min at blood flow rates ranging between 8 and 48 ml/min. This LAD was shown to increase the oxygen saturation level by 25% at the low pressure drop of 29 mm Hg. Finally, a piglet was used to test the gas exchange capacity of the LAD in vivo. The animal experiment results were in accordance with in-vitro results, which shows that the LAD is capable of providing sufficient gas exchange at a blood flow rate of ∼24 ml/min.

An artificial placenta type microfluidic blood oxygenator with double-sided gas transfer microchannels and its integration as a neonatal lung assist device.

Preterm neonates suffering from respiratory distress syndrome require assistive support in the form of mechanical ventilation or extracorporeal membrane oxygenation, which may lead to long-term complications or even death. Here, we describe a high performance artificial placenta type microfluidic oxygenator, termed as a double-sided single oxygenator unit (dsSOU), which combines microwire stainless-steel mesh reinforced gas permeable membranes on both sides of a microchannel network, thereby significantly reducing the diffusional resistance to oxygen uptake as compared to the previous single-sided oxygenator designs. The new oxygenator is designed to be operated in a pumpless manner, perfused solely due to the arterio-venous pressure difference in a neonate and oxygenate blood through exposure directly to ambient atmosphere without any air or oxygen pumping. The best performing dsSOUs showed up to ∼343% improvement in oxygen transfer compared to a single-sided SOU (ssSOU) with the same height. Later, the dsSOUs were optimized and integrated to build a lung assist device (LAD) that could support the oxygenation needs for a 1-2 kg neonate under clinically relevant conditions for the artificial placenta, namely, flow rates ranging from 10 to 60 ml/min and a pressure drop of 10-60 mmHg. The LAD provided an oxygen uptake of 0.78-2.86 ml/min, which corresponded to the increase in oxygen saturation from 57 ± 1% to 93%-100%, under pure oxygen environment. This microfluidic lung assist device combines elegant design with new microfabrication methods to develop a pumpless, microfluidic blood oxygenator that is capable of supporting 30% of the oxygen needs of a pre-term neonate.