3D-Printed Lung-on-a-chip Device for Monitoring of YAP/TAZ Translocation in Murine Lung Epithelial Cells Under Static and Dynamic Conditions

Abstract

Here a lung-on-a-chip device was developed and fabricated using 3D-printing of molds. The lung-on-a-chip device was used as a lung injury model by overstretching of murine lung epithelial cells (MLE-12) monolayers, resembling the injury during mechanical ventilation.

The cells were exposed to 25% strain, representing pathological conditions seen in ventilator induced lung injury of patients with e.g. ARDS. Our results showed a difference in YAP/TAZ activity between the static (no breathing) and dynamic conditions. Moreover, immunofluorescence staining and SEM characterization of the cell layer indicated higher levels of cell loss from the membrane surface when the device was tested under dynamic conditions.

PCL is a synthetic polymer thus it doesn’t have the necessary biological cues for cell attachment, proliferation and differentiation. For this reason, we also aimed to prepare hybrid nanofibers by mixing the synthetic polymer of PCL with a biological component of decellularized lung ECM (dECM). In collaboration with Cellevate, we prepared the electrospun PCL and PCL-dECM hybrid membranes, spun with two different flow rates. The membranes were characterized using Fourier-transform infrared spectroscopy (FTIR), contact angle measurements and scanning electron microscopy (SEM). FTIR spectrums of the membranes confirmed the presence dECM in the PCL membranes. PCL-ECM hybrid membranes showed a reduced hydrophobicity compared to the PCL membrane. SEM PCL-ECM hybrid membranes exhibited a wider mean fiber diameter compared to PCL fibers. The two different PCL-dECM hybrid membranes also exhibited different morphologies, which were fabricated with a lower flow rate, experienced smaller secondary fibers on top of the main fiber structure. While membranes spun with a higher flow rate had uniform fiber structure.

The lung-on-a-chip device presented in this thesis shows promising results to investigate the mechanical stress induce effects on epithelial cells. However, further improvement in the chip design is necessary to enable live-imaging. In this work, we successfully produced the PCL-ECM hybrid membranes. Next, cell studies should be carried out to establish the effects of the dECM incorporation of the material.

Popular Abstract

Microfluidic Device for Modelling Lung injury

Respiratory disease is one of the most common causes of death worldwide. The increase in the prevalence of respiratory diseases is mainly due to cigarette smoking, air pollution as well as bacterial/viral (COVID-19) infections. Despite this increasing frequency, the only option for treatment during end-stage disease is lung transplantation. Thus, development of reliable in-vitro models is necessary for lung disease modelling, regeneration and testing potential therapies.

In respiratory diseases like acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease or COVID-19, breathing ability is impaired that essentially leads patient death. When patients are not able to breathe by themselves, they are connected to the mechanical ventilator. Mechanical ventilator provides enough oxygen to organs. Although it is lifesaving, it can also damage the patient’s lung. Normally, the cells inside the lung are stretched 4-12%. However mechanical ventilation can cause ventilator induced lung injury (VILI) a condition where too much air or too high pressures can cause the alveolar cells to overstretch and eventually die. Patients with ARDS, COPD or COVID-19 for example are especially vulnerable to injury during mechanical ventilation due to their alveoli being exposed to larger levels of stretch.

To model VILI, animal studies have previously been conducted with debatable ethics. With the help of a lung-on-a-chip device VILI can be modeled and reproduced many times and different conditions or drugs can be tested. In this project a lung-on-a-chip device was developed and fabricated using 3D-printing of the molds used for casting the device. This makes the device considerably easier to manufacture compared to many other chips that use advanced microfabrication techniques that are typically used when fabricating modern electronics. In between of the channels of the chip, the fibrous membrane was made of synthetic polymer of polycaprolactone (PCL) membrane were placed. Mouse lung epithelial cells were grown on PCL membrane in the chip. 25 % of cyclic stretch, which resemble the applied stretch during mechanical treatment at a normal breathing rhythm (12 breaths per min) was applied. After the applied stretch, the cell we were shown to die or detach from the membrane to a larger extent than cells that were not stretched. These cells also responded to the stretch on a molecular level. YAP/TAZ a protein inside of the cell translocate to the nuclei when the cell is exposed to stretching and then regulates proliferation, differentiation and much more. This translocation was seen in cells exposed to the stretch but not in the cells that were not stretched, further validating the model.

To make our device as close as possible to the real conditions, we aimed to prepare new hybrid membranes. PCL is a synthetic polymer and it is foreign to lung cells. It doesn’t have any biological cues that the cells like. To overcome this limitation, a hybrid membrane prepared using electrospinning (a technique that forms micro/nanofibers and produces a net-like structure) of the biodegradable polymer PCL and natural lung extracellular matrix (ECM) was characterized to confirm the presence of ECM proteins and fibrous structure of the membranes. Our initial data showed that the lung cells like these membranes and they grew more on this membranes compared to PCL ones.

• author: Rehnberg, Emil
• supervisor: Sinem Tas
• organization: Department of Biomedical Engineering
• course: BMEM01 20201
• year: 2020
• type: H2 - Master's Degree (Two Years)
• subject: Technology and Engineering
• language: English
• id: 9025995
• date added to LUP: 2020-08-14 11:01:52
• date last changed: 2020-08-14 11:01:52