Vascular lung triculture organoid via soluble extracellular matrix suspension

TL;DR: In this article, a viable lung organoid from epithelial, endothelial, and fibroblast stable cell lines in suspension culture supplemented with soluble concentrations of extracellular matrix proteins (ECM).

Abstract: Scaffold-free tissue engineering is desired in creating consistently sized and shaped cell aggregates but has been limited to spheroid-like structure and function, thus restricting its use in accurate disease modeling. Here, we show formation of a viable lung organoid from epithelial, endothelial, and fibroblast stable cell lines in suspension culture supplemented with soluble concentrations of extracellular matrix proteins (ECM). We demonstrate the importance of soluble ECM in organotypic patterning with the emergence of air space-like gas exchange units, formation of branching, perfusable vasculature, and increased 3D growth. Our results show a dependent relationship between enhanced fibronectin fibril assembly and the incorporation of ECM in the organoid. Endothelial branching was found to depend on both soluble ECM and fibroblast. We successfully applied this technology in modeling lung fibrosis via bleomycin induction and test a potential antifibrotic drug in vitro while maintaining fundamental cell-cell interactions in lung tissue. Our human fluorescent lung organoid (hFLO) model accurately represents features of pulmonary fibrosis which were ameliorated by fasudil treatment. We demonstrate a 3D culture method with potential of creating organoids from mature cells, thus opening avenues for disease modeling and regenerative medicine, enhancing understanding of lung cell biology in health and lung disease.

Summary

Introduction

• The extracellular matrix (ECM) is classically known for its roles in cell anchorage and mechanical signaling in organs.
• The use of scaffold-free force aggregated cultures is limited to creating tissue-like structures.
• 11 However, the effects of low, soluble ECM concentrations in organotypic self- patterning and growth have yet to be fully elucidated.
• Here, the authors provide two potential applications of the hFLO: modeling bleomycin-induced pulmonary fibrosis with a pre-clinical therapeutic and a hypoxic angiogenesis study.

Results

• Soluble ECM promotes airspace-like lumen-gas exchange unit formation in suspended aggregates.
• Self-organized structures observed in both gel scaffold and soluble ECM-supplemented cultures include epithelial clustering and an endothelial-fibroblast network, neither of which were observed in the traditional FA culture (0 µg mL-1) (Fig. 1c and Supplementary Fig. 2b-c, 3a-b).
• Together this data confirms that supplementation of soluble Matrigel leads to topographical arrangement of cells similar to those found in native alveoli (Supplementary Fig. 3).
• Here, the authors grew hFLO for 7 days and induced fibrosis using 20 µg mL-1 bleomycin for 3d, after which aggregates were treated with 10 µM fasudil, a ROCK inhibitor for 4 days to observe possible changes in fibrosis (Fig. 5a).
• Additional information could be found in Supplementary Fig. 10.

Discussion

• Until now researchers have struggled to produce pulmonary organoids from stable cell lines.
• The authors define an organoid as a 3D cell aggregate composed of multiple specialized cell types and basement membranes which mimic organ structure and function, in this case, the lung.50 Previous findings in organoid and spheroid research have focused on the role of ECM as a solid anchoring structure.
• 24,25 The authors data show that even in the presence of soluble ECM, endothelial cells do not form networks in the absence of fibroblasts.
• Nevertheless, their findings showing that fasudil ameliorates the features of bleomycin-induced PF in hFLO allows us to study pathogenesis and modulation of PF via ROCK inhibition in a 3-dimensional milieu in vitro.

Conclusions

• In summary, their novel soluble ECM-based 3D culture method promotes organotypic growth using stable mature cells within 14 days of culture.
• The authors believe that this is the first account illustrating that soluble ECM can create an organotypic lung model from mature stable cells.
• Moreover, this lung model has a perfusable vasculature which current lung organoids lack.
• These organotypic features allowed us to model pulmonary fibrosis and demonstrate its resolution using a ROCK inhibitor, fasudil.
• The application of this method in the formation of patient- or primary-derived organoids is yet to be performed, but will be necessary to create high- throughput screens for personalized medicine.

Figures

Fig. 1: Alveolar-like lumen structure formation. a, Representative bright-field images of 3D cell aggregates after 14 days of culture. Scale bars, 300 µm. b, Effect of soluble ECM in aggregate density. Line profile analysis at the major axis of the aggregates expressed as normalized grey value. Lines indicate the grey value range at the specific normalized distance from the center. c, Live 3D fluorescence confocal image (single z-section) showing the selforganized localization of the three cell lines—A549 (green), EAHy (blue), and HFL1 (magenta). In aggregates grown using 300 µg/mL Matrigel, organotypic structures such as the lumina (*) and endothelial networks (arrow) are indicated. Scale bars, 200 µm. d, Hematoxylin and Eosin staining of the aggregates showing that the lumen formation in 300 µg/mL Matrigel aggregates is comparable to the mammalian lung parenchyma. Scale bars, 300 µm for full image; 50 µm for inset. Lumen formation is quantified using number of lumen normalized to aggregate size and percent lumen area. Bar colors are indicated in the micrographs above. Mean, individual measurements, and standard deviation are shown. Three independent cell aggregates and lungs from two mice, one pig were used. **P<0.01, ns (P>0.05) determined by One-way ANOVA with Welch’s correction. e, Fluorescence confocal images (maximum projection) showing localization of epithelial cells (EPCAM, green) and mesenchymal cells (Vimentin, magenta) relative to the lumen. Specific alveolar epithelial subtypes: Type I (Podoplanin, red) and Type II (prosurfactant protein C, blue). Scale bars, 50 µm. f, Transmission electron microscopy of cell aggregates showing stratification and lumen formation in 300 µg/mL Matrigel aggregates. Alveolar type II-like epithelial cells (AT2L) shown with their lamellar body-like inclusions (LBL) and microvilli (mv). Other features include: thin, alveolar type

Fig. 2: Organoid growth and cell survival. a, Bright-field images of 3D cell aggregates during the 14-day culture. Scale bar, 300 µm. b, Line graph showing change in aggregate area. Areas were normalized to their respective day 1 area. Means and standard deviation are shown. Individual line indicates independent experiments performed by independent researchers. N = 10 independently sampled aggregates on each measured day. c, Cell counts at day 14 fold change from day 0 seeding density. Means, individual values, and standard deviation are shown. 3 independent experiments were performed with >3 cell aggregates used; N=12 aggregates. ****P<0.0001 determined by twotailed t-test with Welch’s correction. d, Hematoxylin and Eosin staining of cell aggregates showing stability of lumen structure in hFLO (up to 70 days of culture) as quantified using lumen area and count. Mean, individual values, and standard deviation are shown. ****P<0.0001 determined using two-tailed t-test with Welch’s correction using values from n=27 independent aggregates. e, Cell population composition at days 3, 7, and 14 of culture; n=7 independently sampled aggregates. **P<0.01, ***P<0.001 determined using One-way ANOVA with Welch’s correction comparing day 14 to day 3 values. f, Expression of cleaved caspase 3 of day 14 cell aggregates using imaging flow cytometry. Three independent plates (around 200 cell aggregates) were used and an unstained control from pooled samples was used as a negative gating control. Left bar plot shows percent positive cells with mean, individual points, and standard deviation indicated. Right bar plot shows mean intensities found in each of the samples. ***P<0.001 determined using two-tailed t-test with Welch’s correction.

Fig. 4: Shotgun proteomics. a, Principal component analysis plot of the first and second components. Dots represent the whole proteome of three independent samples used for FA aggregates (grey) and hFLO (pink). Grouping is highlighted using an enclosing oval. b, Volcano plot showing fold changes (log2FC) of hFLO versus FA aggregates. P values were calculated using two-tailed t-tests with Welch’s correction. Red points indicate proteins with P<0.01. c, Distribution of the differentially expressed proteins (DEPs, P<0.01). d, Gene set enrichment analysis (GSEA) plot of all proteins ranked based on enrichment score (Signal2Noise). e, Scatter plot of the top 50 GOTerms enriched in hFLO. Dot size corresponds to number of genes annotated per term and color corresponds to the normalized enrichment score (NES). f, Scatter plot of the top 50 GO-Terms enriched in FA (negative enrichment in hFLO). g, GSEA enrichment plot for selected GO-Terms. Barcodes show individual proteins annotated in each GOTerm and their respective ranks based on enrichment of hFLO versus FA.

Fig. 3: Vascular endothelial branching. a, Live fluorescence confocal images (maximum projection) of day 14 cell aggregates showing EAHy (endothelial, blue) and HFL1 (magenta). Scale bars, 100 µm. b, AngioTool outputs characterizing the abundance and branching of the EAhy-HFL1 networks. We used 8 independent aggregates per condition. **P<0.01, ***P<0.001, ****P<0.0001 determined using two-tailed t-test with Welch’s correction. c, Fluorescence confocal images (maximum projections) of day 14 hFLO showing the presence of vascular cells including endothelial-like (UEA1 lectin), smooth muscle-like (αSMA), and pericyte-like (PDGFRβ) cells. Another perivascular marker, desmin was detected in day 14 hFLO. DAPI was used as the nuclear stain. Scale bars, 100 µm. d, Volumetric analyses of UEA1, αSMA, PDGFRβ, and desmin in FA and hFLO aggregates normalized to DAPI volume. We used n=15 independent cell aggregates for UEA1, αSMA, and PDGFRβ, and n=4 independent cell aggregates for desmin. **P<0.01, ***P<0.001, ****P<0.0001 determined using two-tailed t-test with Welch’s correction. e, Flow cytometric analysis of whole cell aggregates for expression of αSMA, PDGFRβ, and desmin in FA and hFLO. Contour plots show percent gated cells, contours, and outliers were indicated as dots. Three independent plates were used for each. Bar plots show means, individual points, and standard deviation from positive gates. *P<0.05, **P<0.01, ***P<0.001 determined using two-tailed t-test with Welch’s correction.

Fig. 6: Hypoxic vascularization using hFLO. a, Schematic diagram of the workflow showing further vascular growth using hypoxia and pro-angiogenic factors (top left). Bright-field images of aggregates at day 14 of culture (bottom left). vhFLO is hFLO grown under hypoxia for 7 days with supplementation of FGF2 and VEGF. Line profile analysis at the major axis of the aggregates expressed as normalized grey value. Effects of the hypoxic culture in aggregate density is shown using line profile analysis (right). Lines indicate the grey value range at the specific normalized distance from the center. Scale bar, 300 µm. b, Aggregate area at day 14 of culture. 9 independent aggregates were measured for each condition. *P<0.05, **P<0.01 determined using One-way ANOVA with Welch’s t-test. c, HFL1 (red), A549 (green), and EAhy (blue) cell population percent volume composition in hFLO and vhFLO. N=7 independent 3D live confocal images were used. **P<0.01 determined using two-tailed ttest with Welch’s correction. d, Whole aggregate immunostaining of αSMA expression. Scale bar, 200 µm. e, 3D fluorescence confocal image of vhFLO showing vascular-like formation and localization of endothelial-like (UEA1 lectin+, BLUE), pericyte-like (PDGFRβ+, yellow), and smooth muscle-like (αSMA+, red). 3D renderings highlight annular tube morphology typical of a vasculature. Scale bar, 50 µm. f, Illustration of the vascular perfusion assay using dextran 70 kDa flown through a peristaltic pump system (top). The chip used for this assay is shown as a CAD

Full text

1
Vascular lung triculture organoid via soluble extracellular
matrix suspension
Jonard C. Valdoz
1
, Nicholas A. Franks
1
, Collin G. Cribbs
1
, Dallin J. Jacobs
1
, Ethan L. Dodson
1
,
Connor J. Knight
1
, P. Daniel Poulson
1
, Seth R. Garfield
1
, Benjamin C. Johnson
1
, Brandon M.
Hemeyer
1
, Miranda T. Sudo
1
, Jordan A. Saunooke
1
, Mary L. Vallecillo-Zuniga
1
, Matheus
Santos
1
, Brandon Chamberlain
1
, Kenneth A. Christensen
1
, Greg P. Nordin
2
, Ganesh Raghu
3*
,
and Pam M. Van Ry
1*
1
Chemistry and Biochemistry, Brigham Young University, Ezra Taft Benson Building, Campus
Drive, Provo, 84601, UT, U.S.A.
2
Electrical and Computer Engineering Department, Brigham Young University, Provo, Utah,
USA 84602
3
Center for Interstitial Lung Disease, University of Washington 1959, NE Pacific Ave, Seattle,
WA 98195, USA
*correspondence to (email): Pam M Van Ry (pvanry@chem.byu.edu) and Ganesh Raghu
(graghu@uw.edu)
Abstract
Scaffold-free tissue engineering is desired in creating consistently sized and shaped cell
aggregates but has been limited to spheroid-like structure and function, thus restricting its use in
accurate disease modeling. Here, we show formation of a viable lung organoid from epithelial,
endothelial, and fibroblast stable cell lines in suspension culture supplemented with soluble
concentrations of extracellular matrix proteins (ECM). We demonstrate the importance of
soluble ECM in organotypic patterning with the emergence of air space-like gas exchange units,
formation of branching, perfusable vasculature, and increased 3D growth. Our results show a
dependent relationship between enhanced fibronectin fibril assembly and the incorporation of
ECM in the organoid. Endothelial branching was found to depend on both soluble ECM and
fibroblast. We successfully applied this technology in modeling lung fibrosis via bleomycin
induction and test a potential antifibrotic drug in vitro while maintaining fundamental cell-cell
interactions in lung tissue. Our human fluorescent lung organoid (hFLO) model accurately
represents features of pulmonary fibrosis which were ameliorated by fasudil treatment. We
demonstrate a 3D culture method with potential of creating organoids from mature cells, thus
opening avenues for disease modeling and regenerative medicine, enhancing understanding of
lung cell biology in health and lung disease.
Keywords: Extracellular Matrix, Suspension Culture, Lung Organoid, Pulmonary Fibrosis,
Vascularization, Tissue Engineering
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted September 17, 2021. ; https://doi.org/10.1101/2021.09.16.460165doi: bioRxiv preprint

2
Introduction
The extracellular matrix (ECM) is classically known for its roles in cell anchorage and
mechanical signaling in organs.
1
The role of ECM as an anchor is important in modulating
organogenesis,
2,3
cell differentiation,
4
cell survival,
5
and metabolism.
6
These ECM
characteristics have been exploited by tissue engineers to recreate tissue- and organ-like 3-
dimensional (3D) biological materials essential for disease modeling
7,8
and regenerative
medicine.
9
The tunability of ECM stiffness affects stem cell differentiation.
10
However, the roles
of ECM as a soluble supplement are rarely studied and not well defined.
11
The majority of 3D
culture techniques can be broken down into two categories: solid scaffold using some type of
ECM and scaffold free, no ECM.
12
Both culture techniques have advantages and disadvantages.
In tissue engineering, the use of gelled ECM or other solid ECM-like polymers are prevalent in
the construction of organotypic structures.
12
Scaffold-based cultures mimic the presence of a
solid ECM akin to that of native organ tissue.
13
By this, the cells are provided with a substrate for
organotypic attachment leading to biochemical signaling.
1
In several stem cell-based approaches,
cell clusters are embedded in solid ECM scaffolds to encourage organotypic formation.
14
Some
obstacles in solid scaffold-based cultures include slow formation of tissues, inconsistent growth
of aggregates, difficult recovery, and high contamination for downstream analyses.
9,12
Scaffold-free techniques, on the other hand, often use forced aggregation of cells on ultra-low
attachment surfaces and rely on self-patterning of tissue to create consistently sized, reproducible
multicellular aggregates.
9
Due to high initial local cell concentration, self-assembly into tissue-
like structures is relatively faster than scaffold-based methods.
12
This characteristic gives
scaffold-free technique an edge for being used in downstream high-throughput experiments.
However, the use of scaffold-free force aggregated cultures is limited to creating tissue-like
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted September 17, 2021. ; https://doi.org/10.1101/2021.09.16.460165doi: bioRxiv preprint

3
structures. The lack of organotypic structures is attributed to lack of organ-like ECM during self-
patterning.
15
Our hypothesis is that incorporating non-gelling concentrations of ECM proteins will improve
the currently used scaffold-free, suspension culture. This research demonstrates the importance
of the ECM in promoting organotypic patterning, growth, and survival as soluble supplements in
suspension culture. Previous studies show that the composite properties of ECM are capable of
determining stem cell fate.
16
Additionally, soluble ECM enhances cell proliferation and survival
in 2D mesenchymal cell cultures, and enhanced formation of tight junctions in hepatic spheroid
cultures.
11
However, the effects of low, soluble ECM concentrations in organotypic self-
patterning and growth have yet to be fully elucidated. We illustrate the use of soluble ECM in the
development of an organoid from stable cells within a 14-day time period. We now refer to it as
human fluorescent lung organoid (hFLO). hFLO has airspace-like lumen-gas exchange units and
a perfusable vasculature, not present in traditional scaffold-free spheroids.
Here, we provide two potential applications of the hFLO: modeling bleomycin-induced
pulmonary fibrosis with a pre-clinical therapeutic and a hypoxic angiogenesis study. Pulmonary
fibrosis (PF) in humans is a progressive manifestation of interstitial lung diseases of known and
unknown cause and leads to distorted lung parenchyma, irreversible pulmonary fibrosis,
honeycomb lung, respiratory failure and death.
17
While several agents are being investigated in
the clinic to determine safety and efficacy of pharmacologic agents (clinicaltrials.gov), only two
drugs have been approved for clinical use. These antifibrotic agents, nintedanib and pirfenidone
are currently used in clinical practice but merely slow down disease process.
18
A long standing
and well accepted model used to understand pathogenesis and modulation of PF is the bleomycin
induced injury model.
29
Here, we show that with bleomycin induction, the hFLO is a viable
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
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4
model in mimicking PF features in vitro and could be applied in a preclinical drug trial of an
antifibrotic drug.
Results
Soluble ECM promotes airspace-like lumen-gas exchange unit formation in suspended
aggregates. Previous studies have used soluble matrix proteins as media additives to improve
aggregation
19,20
and as a potent pro-proliferative factor.
11
Despite this, most research has focused
on the effects of ECM stiffness and stem cell fate. The relationship between soluble ECM and
self-patterning towards 3D organotypic growth is yet to be discovered. To model the lung
alveolus, we used traditional forced aggregation (FA) method and added varying types of soluble
concentration of ECM as a supplement. In order to visualize topographical changes
accompanying ECM changes, three fluorescently labeled stable cell lines were employed to
represent the three major alveolar tissue types including endothelial EA.hy926 (EAhy, Azurite-
tagged), lung epithelial type II-like A549 (eGFP-tagged), and normal lung fibroblast HFL1
(mCherry-tagged) (Supplementary Fig. 1a-c). A549 is a cancer cell line with increased
expression of mesenchymal, pro-metastatic proteins.
21
To mitigate these traits, we used
fluorescence-activated cell sorting (FACS) to deplete metastatic N-Cadherin
high
population which
selects for the more stable epithelial cell population.
22
We then engineered a 3D triculture to model the lung alveolus and the morphological changes
accompanying soluble ECM concentration. In the native lung alveolus, the epithelium forms
airspaces with interstitial and vascular cells between the epithelium. We incorporated labeled or
unlabeled A549-eGFP, HFL1-mCherry, and EAhy-Azurite in 3D tricultures with varying ECM
concentrations—high (gel scaffold, ≥3 mg ml
-1
), low (soluble ECM-supplemented suspension,
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted September 17, 2021. ; https://doi.org/10.1101/2021.09.16.460165doi: bioRxiv preprint

5
≤300 µg ml
-1
), and no ECM (traditional FA) (Fig. 1 and Supplementary Fig. 2). Cell seeding
ratios were followed based on endogenous human lung cell composition which we determined to
be roughly 1:2:2 (epithelial: interstitial: vascular) (Table S1).
23
With this ratio, high ECM
concentrations in gel scaffolds led to the formation of multiple A549 epithelial clusters without
cohesion of all three cell lines (Supplementary Fig. 2a-b). In gel scaffolds, fibroblast-endothelial
(HFL1-EAhy) network formation were observed as reported by previous researchers
24,25
(Supplementary Fig. 2b). In comparison, all soluble ECM concentrations tested led to the
formation of a singular cell cluster with all three cell lines incorporated (Fig. 1a). An important
component of the human alveolus is alveolar sacs, which are small airspace-filled voids made
from epithelial cell and separated by a common septum. Bright-field images of cultures with
soluble ECM were analyzed for possible less dense airspace-like voids. Images and grey line
profile analysis show several less dense regions with supplementation of 300 µg ml
-1
Matrigel,
while traditional FA cultures appear as densely packed aggregates (Fig. 1b). Live confocal
imaging reveals self-organized structures in the triculture aggregates similar to those observed in
mammalian alveolus (Fig. 1c and Supplementary Fig. 2b-c). Self-organized structures observed
in both gel scaffold and soluble ECM-supplemented cultures include epithelial clustering and an
endothelial-fibroblast network, neither of which were observed in the traditional FA culture (0
µg mL
-1
) (Fig. 1c and Supplementary Fig. 2b-c, 3a-b). In soluble 100 µg ml
-1
collagen
suspension cultures, epithelial cells form a dense core aggregate; however, 300 µg ml
-1
Matrigel
cultures form airspace-like lumina (Fig. 1a-c and Supplementary Fig. 2c, 3a-b).
13-16
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted September 17, 2021. ; https://doi.org/10.1101/2021.09.16.460165doi: bioRxiv preprint

Frequently asked questions

Q1. What are the contributions mentioned in the paper "Vascular lung triculture organoid via soluble extracellular matrix suspension" ?

Here, the authors show formation of a viable lung organoid from epithelial, endothelial, and fibroblast stable cell lines in suspension culture supplemented with soluble concentrations of extracellular matrix proteins ( ECM ). The authors demonstrate the importance of soluble ECM in organotypic patterning with the emergence of air space-like gas exchange units, formation of branching, perfusable vasculature, and increased 3D growth. The authors demonstrate a 3D culture method with potential of creating organoids from mature cells, thus opening avenues for disease modeling and regenerative medicine, enhancing understanding of lung cell biology in health and lung disease. The authors successfully applied this technology in modeling lung fibrosis via bleomycin induction and test a potential antifibrotic drug in vitro while maintaining fundamental cell-cell interactions in lung tissue. 

Q2. What have the authors stated for future works in "Vascular lung triculture organoid via soluble extracellular matrix suspension" ?

These features also make hFLO a feasible and important tool for time- sensitive disease modeling like the COVID-19 pandemic or future respiratory pandemics. Because hFLO is a human in vitro model, it has the potential to replicate disease pathogenesis and drug interactions that current animal models are unable to show. 

Q3. What are the hallmark features of a normal interstitial pneumonia?

58Histopathological and biochemical patterns of usual interstitial pneumonia (UIP), the hallmarkfeature of IPF include: epithelial injury leading to disrupted basement membrane (ie. increasedcollagen deposition), loss of airspaces, presence of dominant fibroblastic foci, and increasedmesenchymal markers. 

Q4. What are some of the problems in solid scaffold-based cultures?

14 Someobstacles in solid scaffold-based cultures include slow formation of tissues, inconsistent growthof aggregates, difficult recovery, and high contamination for downstream analyses. 

Q5. What is the role of the vascular unit in the development of lung organoids?

The authors posit thatthe use of this basic vascular unit is vital in the improvement of current epithelial organoids inexpanding their growth and stability. 

Q6. What are the common 3Dculture techniques?

11 The majority of 3Dculture techniques can be broken down into two categories: solid scaffold using some type ofECM and scaffold free, no ECM. 

Q7. What are the structures observed in both gel scaffold and soluble ECM cultures?

Self-organized structures observedin both gel scaffold and soluble ECM-supplemented cultures include epithelial clustering and anendothelial-fibroblast network, neither of which were observed in the traditional FA culture (0µg mL-1) (Fig. 1c and Supplementary Fig. 2b-c, 3a-b). 

Q8. What is the significance of the clustering of protein-protein correlation across allsamples?

Hierarchical clustering of protein-protein correlation across allsamples shows the general proteome landscape changes and implies co-regulation amongproteins associated with hFLO (Supplementary Fig. 8g). 

Q9. What are the two antifibrotic agents currently used in clinical practice?

These antifibrotic agents, nintedanib and pirfenidoneare currently used in clinical practice but merely slow down disease process. 

Q10. How did the authors measure the size of FA and hFLO?

The authors trackedaggregate size using bright-field microscopy during a 14-day culture of both FA and hFLO andfound that hFLO aggregates increase 2-fold in size while FA aggregates remained nearlyconstant (Fig. 2a-b). 

Q11. What is the role of the soluble Matrigel in the formation of fibrillar structures?

Their data suggest that the soluble Matrigel is being incorporated into theorganoid which then magnifies the assembly of fibronectin into fibrillar structures.