Bioengineering lung tissue using extracellular matrix based 3D bioprinting

Project Information

3DBIOLUNG

Grant agreement ID: 805361

DOI

10.3030/805361

Project closed

EC signature date 10 December 2018

Start date 1 January 2019

End date 31 December 2023

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 1 499 975,00

EU contribution

€ 1 499 975,00

Coordinated by

LUNDS UNIVERSITET
Sweden

Periodic Reporting for period 4 - 3DBIOLUNG (Bioengineering lung tissue using extracellular matrix based 3D bioprinting)

Reporting period: 2023-07-01 to 2023-12-31

Summary of the context and overall objectives of the project

Immense progress has been made in the development of tissue engineered therapies for many diseases, but there is still a general lack of scalable, precise and controllable, manufacturing techniques to produce human tissue constructs for transplantation or surgical reconstruction. 3D printing is one such potential manufacturing technique; while 3D printing has rapidly evolved, progress with 3D bioprinting (when cells are included) has been slower due to a lack of bioinks which are tissue specific and that can produce constructs with the precision needed for functional tissue. Ideal bioinks should have the necessary rheological properties to be compatible with 3D bioprinting as well as biologically and mechanically support the development of mature tissue ex vivo (prior to transplantation) as well as in vivo (after transplant, to allow integration with the host vasculature without immune rejection). The overall aim of this project was to develop a new bioink which is tissue specific and can be used with extrusion bioprinting to support the development of translationally relevant, 3D transplantable tissue constructs. In general, there is a chronic shortage of donor organs for all tissues, but particularly for lungs where there is only around 6000 lung transplants performed each year.
We have developed a new class of bioinks by combining enzymatically digested and tissue-specific decellularized lung extracellular matrix (dECM) with alginate. Inclusion of the dECM in sodium alginate bioinks improved cell viability, conveyed shear thinning properties to the bioinks, and reduced the foreign body response to implanted cell free, 3D bioprinted constructs. We also established multiple new imaging techniques which enable non-destructive imaging of intact bioengineered constructs, including both label dependent and label independent approaches which can be used in live and fixed approaches using confocal fluorescence microscopy, light sheet fluorescence microscopy (LSFM), and optical photothermal infrared spectroscopy (OPTIR).

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

We have developed techniques to generate a novel bioink which allows for customization for each tissue type through optimization of the combination of a naturally derived material from seaweed currently used as a biomaterial in clinical applications, alginate, with proteins derived from acellular tissue scaffolds which have been mechanically and enzymatically digested. We used a custom-built dual-extrusion 3D bioprinter from a commercially available thermoplastic printer and validated that our bioinks can be used to 3D bioprint alginate and alginate-extracellular matrix hydrogels in the forms of tubes, discs, and branching structures. This has allowed use to demonstrate that both components of the bioink are necessary to support cell lines as well as primary human lung cells during 3D bioprinting as well as during the tissue maturation process ex vivo (De Santis et al. Adv Materials 2021).
To evaluate 3D bioprinted constructs, we developed tracking strategies using fluorescence microscopy, live/dead staining, and live nuclear dyes to assess cell survival (apoptosis versus necrosis), nuclei count, and nuclear morphology over time (Nowakowska et al. AJP-Lung 2024). We physically characterized the bioprinted constructs using rheology and wire myography as well as developed techniques to image these constructs with standard histology, confocal imaging, LSFM as well as scanning electron microscopy (Da Silva et al. Front Biomat Sci 2023). This has been applied to both living and fixed ex vivo tissue engineered constructs as well as those which were transplanted into animal models mimicking solid organ transplantation (De Santis et al. Adv Materials 2021). Hybrid bioinks are able to regulate angiogenesis in both a chick embryo chorioallantoic membrane (CAM) assay and through a full thickness implant (evaluated using LSFM) as well as suppress a negative immune response through polarizing immune cells which infiltrate the 3D bioprinted construct.. For the imaging of larger constructs, this required the development of new techniques for fixation, dehydration, delipidation and optical clearing of samples to enable preservation of 3D bioprinted constructs’ architecture (De Santis et al. Adv Materials 2021). We also established workflows for visualizing and quantitatively evaluating both living and fixed constructs in 3D, including visualization and segmentation in virtual reality.
During the action, the first large scale public atlases of the human lung and primary human lung epithelial cells cultured ex vivo became available. We developed bioinformatic techniques to perform computational deconvolution of bulk RNA-sequencing data of our bioengineered constructs with these datasets to better understand how the ex vivo environment created by bioinks impacts cellular states and how close our constructs are to native, healthy in vivo tissue.
The PI and team members have also distributed their work in diverse public formats such as more than 50 local, national and scientific conferences, podcasts (https://stemcellpodcast.com/tag/darcy-wagner) and webinars (https://www.youtube.com/watch?v=vGJQHXNXRyQ).

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

We developed significant knowledge regarding the properties needed in novel bioinks which contain tissue specific cues. We developed and applied several state of the art characterization techniques to 3D bioprinted constructs for the first time. In particular, we developed the capability to optically clear transplanted constructs which enabled us to image entire, intact constructs using light based imaging which has previously not been possible. This allowed us to examine the 3D vasculature network which formed upon implantation of 3D printed constructs using our bioinks into both immunocompetent and immunodeficient animals (mimicking transplantation). We also demonstrated proof-of-principle for live imaging using co-linear infrared excitation coupled to visible light based detection in hydrated samples using OPTIR to identify proteins and lipids in living, hydrated samples containing polymers, extracellular matrix and cells (Gvazava et al. J Am Chem Soc 2023). This included serial imaging of the same living sample over time. As water is known to absorb infrared light in the same wavenumber region as proteins, this is a major advance with implications far beyond 3D bioengineering of tissues and could transform characterization of tissues using non-invasive and label-free imaging of intact 3D constructs.
Our bioink can be used in dual-extrusion approaches to produce layered airways with regionally specified primary human airway cells, which is a needed advance for creating transplantable constructs with tissue in the appropriate orientation. Bioprinted small human airways can also be used as new models of airway disease (e.g. infection) to better understand disease or model potential therapies.
We have begun work to scale up our approach to bigger constructs by expanding cells in rotational bioreactors and to improve the long term mechanical stability of 3D bioprinted airways through the further development and refinement of our bioinks using dual crosslinking approaches (Petrou et al. J Mat Chem B 2020 and unpublished. We have made significant advances of bioreactor design using 3D printing approaches as well as rotational bioreactors to improve diffusion under low shear flow rates to enable longer term culture of bioengineered constructs.

3D bioprinted airway