Cian O'Leary and his lab are developing 3D bioengineered in vitro models of the lung and other tissues using scaffolds. [1] They have created bilayered collagen-hyaluronate scaffolds that support a mucociliary epithelial phenotype in lung cell culture models. [2] The lab is also working on 3D hydrogel models of pancreatic cancer to study cell-matrix interactions and cancer progression. [3] Future work includes developing dynamically stiffening hydrogel models and applying these platforms to study lung cancer and the pre-metastatic niche.

1. Bioengineered 3D Co-culture Lung In
Vitro Models: Platforms to Integrate
Cell-Matrix Interactions
Cian O’Leary PhD MPSI
School of Pharmacy and Biomolecular Sciences
Royal College of Surgeons in Ireland
cianoleary@rcsi.ie
@cian_o_leary

2. Conditions of Interest
The respiratory system: Idiopathic pulmonary fibrosis (IPF)
Cancer: Lung (NSCLC), Pancreatic (PDAC)
Soft tissue injury: Tracheal repair, Tympanic membrane repair
O’Leary Lab: Research Themes
Formulation &
dosage form
development
Druggable targets
with novel agents or
repurposed drugs
Novel disease models
of Cell-ECM
microenvironments
atRA
3DP Tubular
Biomaterials
Drug-releasing
scaffolds
Inhalable Particulate
Formulations

3. Challenges with Preclinical Animal Models in Disease Modelling & Drug Discovery
O’Leary C, et al. (2015). Tissue Eng Part B Rev.
• Physiological differences
• Therapeutic responses
• Toxicological responses
• Presence or absence of disease in species
• Anatomical and histological differences
• Presence or absence of organs
• Cell types and cell distribution
• Tissue architecture
• Dosing considerations and pharmacokinetics
• Method of administration
• Limited volumes for dose administration
• Sample collection
• Ethical and economic considerations
• The “3Rs”: Reduce, refine, replace
• Housing costs

4. Bioengineering & The Tissue Engineering Triad

5. The Limitations of Classical Respiratory In Vitro Cell Models
Klein SG, et al. (2011). Toxicol In Vitro.
ALI
Biomaterial platforms have the potential to improve current
in vitro models

6. Bioengineered Tracheobronchial Scaffolds as 3D In vitro Models
• Method of CHyA-B scaffold fabrication
– Modification of fully-porous collagen-glycosaminoglycan scaffolds previously
developed by TERG1, 2
– Bilayered structure: (i) Dense film top-layer and (ii) porous sub-layer
– Composition reflective of native tracheobronchial ECM: Collagen, hyaluronic acid
Collagen-GAG Scaffolds
1. O’Brien FJ, et al. (2004). Biomaterials.
2. Haugh MG, et al. (2010). Tissue Eng Part C Methods.
CHyA-B:
Bilayered Collagen-Hyaluronate Co-Polymer
Objectives
1. Design a bioengineered scaffold ECM analogues for respiratory tissue
2. Establish a respiratory 3D co-culture platform using this scaffold
3. Assess epithelial cell functionality in this co-culture platform

7. Bilayered Scaffolds:
Successful fabrication & Key Manufacture Parameters Identified
Anneal Cycle:
Mean Pore Diameter 80µm
Tf -10: Mean Pore Diameter 70µm
O’Leary C, et al. (2016). Biomaterials.

8. Objective:
Respiratory Co-Culture & Epithelial Analysis

9. Respiratory Mono-Culture:
CHyA-B Scaffolds supported a Mucociliary Epithelial Phenotype Vs Cell Inserts
O’Leary C, et al. (2016). Biomaterials.
CHyA-B

10. Respiratory Mono-Culture:
CHyA-B Scaffolds supported a Mucociliary Epithelial Phenotype Vs Cell Inserts
O’Leary C, et al. (2016). Biomaterials.
Cell
Insert
CHyA-B
ZO-1 TEM
MUC5AC

11. Primary Tracheobronchial Epithelial Cell Co-Culture:
Mucociliary Phenotype with Barrier Function on CHyA-B Scaffolds
Cell
Insert
CHyA-B
Monoculture Co-culture
Yellow: β-Tubulin IV ; Red: F-Actin; Green: ZO-1
CHyA-B
Co-culture
Physiological Range

12. O’Leary Lab: Ongoing Research with CHyA-B
Tehreem Khalid
Luis Soriano
Soriano L, Khalid T, et al. (2021). Biomedicines.
Soriano L, Khalid T, et al. (2021). Eur Respir Rev.

13. Where do we go from here with Cancer Models?
Huang J, et al. (2021). Signal Transduct Target Ther.
Chitty JL, et al. (2018). F1000 Res.

14. Cell-Matrix Interactions: Fundamental Concepts of Mechanotransduction
1. Almouemen N, Kelly HM, O’Leary C (2019). Comput Struct Biotechnol.
2. Humphrey JD, et al. (2014). Nat Rev Mol Cell Biol.
• Cells respond to a combination of biochemical and
biomechanical stimuli within their physiological
microenvironment:1
• Biophysical stimuli from the extracellular matrix (ECM) are
mediated by matrix composition & mechanical properties.2
• Different types of matrix proteins can be recognised by cell
receptors to transduce different signals:
• Matrix proteins include families of collagens, proteoglycans,
laminins, fibronectin…
• Cell receptors include integrins, CD44, discoidin domain-
containing receptors…
• Cell responses are also affected by mechanical stiffness of
these matrix substrates:
• Stiffness relates to the matrix’s resistance to deformation.
• Effector responses to cell ligand density and matrix elasticity
include differentiation, migration, and disease progression.

15. Matrix Stiffness & Pathophysiology
1. Guimarães CF, et al. (2020). Nat Rev Mater.
2. Huang J, et al. (2021). Signal Transduct Target Ther.

16. The Development of a Tissue Engineered 3D In Vitro Model of Pancreatic Cancer
1. Dong Z, et al. (2019) RSC Adv.
2. Almouemen N. (2020) MSc Thesis.
Nour Almouemen
Thesis Objectives
1. Develop a reproducible hydrogel-based 3D biomaterial with PDAC-relevant
mechanical properties for in vitro applications.
2. Examine the suitability of the 3D hydrogel biomaterial for monoculture and
co-culture studies.
GelMA
Gelatin

17. Fabrication of a series of GelMA Biomaterial Substrates with Different Stiffness
GelMA:
Gelatin:
DoF = 32%

18. Cancer Cell Mono-Culture on GelMA Biomaterials
Day 2 Day 5 Day 7
0
1×104
2×104
3×104
4×104
Time (days)
DNA
concentration
(ng/ml)
1.5%
3%
5%
✱
✱✱
✱✱✱
c)
b)
Day 2 Day 5 Day 7
0
1
2
3
Time (days)
1.5%
3%
5%
Relative
Fluorescence
ns
1.5% 3% 5%
0
10
20
30
Relative
Area
Coverage
%
1.5%
3%
5%
✱✱✱
✱✱
a)
d)
1.5% 3% 5%

19. Pancreatic Stellate Cell Mono-Culture on GelMA Biomaterials
Day 2 Day 5 Day 7
0.0
0.5
1.0
1.5
Time (days)
Relative
flourescence
1.5%
3%
5%
✱✱✱
✱✱✱
Day2 Day5 Day7
0
2×105
4×105
6×105
Time (days)
DNA
concentration
(ng/ml)
1.5%
3%
5%
✱✱✱
✱✱✱
c)
a)
d)
1.5% 3% 5%
0
20
40
60
80
100
Relative
Area
Coverage
%
1.5%
3%
5%
✱✱✱
1.5% 3% 5%
b)

20. Cancer Co-Culture on GelMA Biomaterials
c) d)
a)
1.5% 3% 5%
0
20
40
60
80
100
GelMA Concentration
Relative
Area
Coverage
%
1.5%
3%
5%
✱✱✱
1.5% 3% 5%
b)

21. Towards Dynamically-Stiffening GelMA Substrates
Mark Lemoine
Project Objectives
1. Develop a GelMA Biomaterial with greater stiffening capacity.
2. Develop a GelMA Biomaterial with the capacity to stiffen independent of
substrate concentration.
GelMA
Gelatin
Project Questions
1. Can we decouple ligand density and stiffness with this
biomaterial system?
2. What are the GelMA properties that will facilitate this?
Hypotheses
• Increasing DoF could increase crosslinking capacity and in
turn, GelMA stiffness.
• Higher GelMA concentration ≥ 5% could also increase
crosslinking & stiffness.
• Increasing photoinitiator concentration could increase
crosslinking & stiffness.

22. Towards Dynamically-Stiffening GelMA Substrates
0
5
10
15
20
25
30
35
40
45
0.017mM +0.017mM +0.054mM +0.204mM +0.204+0.34mM
Compressive
modulus
(kPa)
LAP crosslinking concentration
GelMA 10%: Stepwise Crosslinking
0
10
20
30
40
50
60
70
80
90
1.5 3 5 10
Compressive
modulus
(kPa)
Concentration (%w/v)
GelMA Low DoF Vs High DoF Batch 0 and Batch 1
Dynamic Capacity
• One substrate can be repeatedly stiffened.
• Cultured cells can be cultured in one substrate and experience gradual
stiffening of microenvironment to resemble disease progression.
• Greater stiffness ranges can be achieved.

23. IRC PhD 2021: Lung Cancer Bioengineering & The Pre-Metastatic Niche

24. The ILCA Community
• Bioengineered models
• Primary Samples/Personalised Samples
• Immunological Components of TME
• Platforms for large-scale analysis/Bioinformatics
• Drug Development/Testing
• Stromal modulation
• Understanding Adverse Drug Reactions
• Mechanism of Action Studies/Phenotypic Pharmacology

25. Acknowledgements
O’Leary Lab
Nour Almouemen
Joanne Reardon
Lee Sherlock
Tehreem Khalid
Vera Almeida
Luis Soriano
Dr Mark Lemoine
Dr Sonia Gera
Collaborators
Prof Sally-Ann Cryan
Prof Fergal O’Brien
Prof Helena Kelly
Prof Killian Hurley
Dr Graeme Kelly
Funding
Enterprise Ireland (EI)
European Molecular Biology Organization (EMBO)
Fulbright Commission
Irish Lung Fibrosis Association (ILFA)
Irish Research Council (IRC)
Ministry of Kuwait
SFI-AMBER Research Centre
SFI-CÚRAM Research Centre