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Development of an innovative Airway-on-Chip microbiome cell culture model to investigate host-microbiome interactions at the airway epithelial surface

Periodic Reporting for period 2 - EpiCBiome (Development of an innovative Airway-on-Chip microbiome cell culture model to investigate host-microbiome interactions at the airway epithelial surface)

Okres sprawozdawczy: 2019-03-01 do 2020-02-29

Background: Chronic Obstructive Pulmonary Disease (COPD) is a progressive chronic lung disease characterized by development of irreversible loss of lung function and persistent lung inflammation. In addition, patients with COPD have a higher frequency of respiratory infections, which are considered to be main triggers for exacerbations (“lung attacks”). Exacerbations accelerate disease progression, reduce quality-of-life and are associated with substantial mortality. Alarmingly, today COPD represents the third cause of death worldwide. These dramatic statistics underscore the need to better understand COPD in order to identify new and effective therapeutic strategies. One such strategy may lie in targeting the airway microbiota. Similar to the gut, the airways also harbour a variety of microorganisms that have a unique composition called the airway microbiota. Research on the gut revealed that disturbances in the gut microbiota composition could be linked to disease. Much less is known about the contribution of airway microbiota to health and disease. Interestingly, in patients with chronic inflammatory lung diseases the composition of the airway microbiota has changed. However, it is currently unclear if and how these changes contribute to an increased susceptibility for respiratory infections in these patients. Since this research question is extremely difficult to study in human lungs, we need cell culture models that represent the lungs as accurate as possible and include microbial co-culture.

Original aim and objectives: This proposal is focused on the airway epithelium, a layer that lines the airways and plays a central role in protection against infection and regulating inflammation. The project aimed to develop an innovative human state-of-the-art Airway Lung-Chip microbiota co-culture model and use this model to investigate the interaction of the airway epithelium with the microbiota in a healthy and diseased environment and investigate how these interactions contribute to susceptibility for infection. This knowledge will be of significant importance to elucidate the mechanisms that underlie the observed changes in airway epithelial composition and airway microbiota composition in patients with COPD. This knowledge could be applied to future research efforts that will contribute to the development of more effective healthcare strategies for COPD.

Conclusions: Optimized protocols for co-culturing airway epithelial cells and endothelial cells in the Airway Lung-Chip have been developed, however not in co-culture with microbes yet. In addition, the researcher obtained extensive training, skills and expertise in Organ-Chip technology. This chip platform is now operational in the laboratory of the researcher, and therefore this final part of the optimization will be anticipated in the near future. Importantly, a co-culture model was developed on Transwells (a less complex model, in contrast to chips) with human airway epithelial cells and microbiota. Results from explorative experiments in this model showed that impairing differentiation of the airway epithelium significantly changes the interaction with the microbiota: cell cultures of which the airway epithelium had an altered (diseased) composition, displayed higher numbers of bacteria, quicker outgrowth and higher levels of pro-inflammatory mediator production.
Taken together, this ambitious project has shown an important proof-of-principle for the development of complex microbiota-containing lung epithelial cell cultures. This is important because both the Organ-Chip field and the airway microbiota field are young developing research fields. In addition, the project has advanced the career of the researcher to an independent, tenured position, allowing further expansion of the developed research lines.
The full first year of the granted period was spent by the researcher at the Organ-Chip company Emulate Inc. who are located in Boston, MA, U.S.A. to obtain hands-on experience with their Organ-Chip platform. At Emulate, the researcher got training and developed routines and expertise by working with the Airway Lung-Chip and several pilot experiments were performed to assess the complexity of developing a protocol for a stable long-term co-culture between airway epithelial cells and microbiota. After one year, Emulate’s Organ-Chip platform was installed in the Department of Pulmonology at the Leiden University Medical Center to continue the studies in the Netherlands. The second year of the granted period focused on finalizing the protocol for the Airway Lung-Chip and additionally performing experiments on Transwells of microbiota co-cultured with healthy airway epithelium and with cultures in which differentiation of the epithelium was modulated, representing diseased lung epithelium. Results from the first explorative studies showed that ‘diseased’ epithelium was more susceptible for microbiota inoculation, had a quicker outgrowth and gave higher pro-inflammatory responses. Large donor variation was also observed between airway epithelial cell donors and their responses to the inoculated microbiota, which is in line with the hypothesis that indeed the airway epithelium is a central factor in microbiota control. Exploitable results from this project are the protocols for the Airway Lung-Chip culture that have been optimized. These will be used by Emulate Inc. after publication by the LUMC to distribute to their customer base. Currently there are no further exploitable results for the LUMC in this project. Results will be disseminated via publications in international peer-reviewed scientific journals, via invited lectures and at (inter)national conferences. In addition, we have regularly addressed our work on social media, via magazines, such as Lab Vision and internal websites of the LUMC. We have also discussed our research with patients and general public via the LUMC Science day, a Dutch Lung Foundation-organized meeting for patients (Longpunt) and by collaborations with for example the Dutch organization for the advancement of animal-free research.
Currently we have developed a protocol for bronchial airway epithelial cell cultures on the commercial Organ-Chip platform from Emulate. In the future we expect to extend this protocol with a microbial co-culture. In addition, we have developed a Transwell model of microbial co-culture with airway epithelial cells and this model provides a further tool to support studies on host-microbe interactions in the lungs. We hope by publishing these protocols that the microbiota research field can be supported with new research initiatives. Therefore, the immediate impact that we expect from our data are mainly on the follow-up of the use of our protocols by the scientific community. In addition, the impact on the career of the researcher from this protocol was significant as the gain in expertise has supported her research career independence as she obtained a permanent contract. Our research with use of these models will continue and will further highlight how altered airway epithelial differentiation affects microbiota levels and composition, as our current data suggest that this is a contributing factor.
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