77 Innovative 3D models for understanding mechanisms underlying lung diseases: powerful tools for translational research PRECISION CUT LUNG SLICES (PCLS) PCLS are a unique translational ex vivo model of peripheral lung tissue that have applicability for many different research approaches including drug discovery and toxicity screenings [11, 12]. The slice lung model was first introduced as a pharmacological model by Dandurand et al. [13]. Unlike tissue fragments obtained by cutting or shredding explanted tumour resections [14], PCLS are well-defined models with standardised protocols for preparation and use in experiments. Upon collection, the lungs are filled via the trachea in animals or bronchi/bronchioles in human lung explants with a low melting agarose solution (0.75% - 3%) and then cut into slices of 200 - 500 μm thickness [15, 16]. The slices consist of distal airways and vessels with their intact lung architecture, maintaining cell-cell interactions and ECM. It is possible to sequentially study various lung cell populations in airways and vessels of different sizes and anatomical locations from a healthy or diseased lung, thus optimally utilizing available resources. Such an approach provides opportunities for specialised screening of the responses of alveoli, bronchioles, vessels, and surrounding ECM. Calcium fluxes and ciliary beat frequency can be followed and visualised in the small airways [16, 17], with smaller airways being known to respond to a greater extent to bronchoconstrictors than larger airways [15, 18]. In some instances, the thickness of the slices may be a concern as the readouts and functionality can differ depending on the thickness. Therefore, it is important to describe slice thickness, area of the slices, and lumen size of airways and pulmonary vessels when reporting generated data. Moreover, cutting and embedding the lung tissues in agarose can trigger repair and regenerative responses that can subsequently affect further experiments. Thorough washing and resting periods prior to experimental procedures can considerably reduce the effect of the processing steps. The viable culture period (5-28 days) is dependent on the type of analysis to be performed and should always be specified to enable replication of the studies. Cryopreservation of PCLS is a way forward to maximise the number of slices that can be used per lung and the possibility to share valuable material, however current protocols under development for cryopreservation need further improvement to enhance PCLS viability and functionality upon revival. PCLS can also be modified to mimic disease with different approaches and protocols either by treating the animal prior to harvesting of the lung or by directly treating the PCLS. Examples of applications of such models include allergic asthma [19, 20], emphysema [21, 22], acute respiratory distress syndrome (ARDS) [23], fibrosis [24, 25]. PCLS can also be decellularised and used as lung scaffolds with native ECM architecture for 3D cell culture studies with and without cyclic stretch [26-28]. Material availability and viable culture duration limits the use of PCLS, however, these models provide unmatched 4
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