MASSIMO PINZANI, MD, PhD, FRCP, FAASLDSheila Sherlock Chair of Hepatology

UCL Institute for Liver and Digestive Health

Royal Free Hospital, London, UK

m.pinzani@ucl.ac.uk

www.ucl.ac.uk/medicine/liver-and-digestive-health

LIVER TISSUE ENGINEERING: WHERE

ARE WE NOW?

Disclosures (2019)

1. Inventor and patent holder ELF test (Siemens)

2. Speakers bureau: Echosens (Paris, France)

3. SAB/Consultancy: Promethera (Belgium); NeuroVive (Sweden);

Chemomab (Israel); Median Technology (France/USA); Boheringer-Ingelheim

(Germany); Takeda (USA)

4. Co-Founder and Director, Engitix Ltd (UCL Spin-out) (UK)*

5. Co-Founder and Director, 3P-Sense Ltd (UCL Spin-out) (UK)**

6. CMO, Hepatotargets Ltd (Cambridge University Spin-out) (UK)***

6. Educational Councillor EASL Governing Board (2015-2019)

7. Chair EASL Consortium for Regenerative Hepatology (2019-2022)

* Regenerative medicine and tissue engineering

** Nanotechnology diagnostics

*** Liver Cell therapy

From Discovery to Clinical Applications

Drug target discovery based on 2D cell

cultures on plastic

Expansion and validation in animal models of chronic liver injury: no model is able to reproduce

human pathophysiology

No translation into clinical trials and very high failure rate in the trials so far performed (>95%)

No licensed antifibrotic drugs after more than 40 years of active research (the case of liver fibrosis)

Wrong targets? Wrong validation methodology?

Pathways of Stellate Cell Activation in Liver Injury

Changes in ECM quantity and relative composition

Altered MATRISOME

Need to focus on mechanisms and preclinical models easier to translate into clinical applications:

The fibrotic microenvironment :a. Hypoxia and neo-angiogenesisb. Anaerobic metabolism (e.g. lactate)c. 3D in vitro modelsd. Hepatic matrisome and matrikinese. Tissue stiffness and contraction

Understanding Liver Fibrosis

3D Models to Study Liver Pathophysiology

1. – Precision-Cut Liver Slices (PCLS)

2. – Spheroids and Organoids

3. – Microfluidics and Microphysiology Systems (Lab-on-a-chip)

4. – Liver ECM engineered 3D scaffolds

5. – Liver ECM bio-printed last generation organoids

Tissue Engineering

3D Models to Study Liver Pathophysiology

1. – Precision-Cut Liver Slices (PCLS)

2. – Spheroids and Organoids

3. – Microfluidics and Microphysiology Systems (Lab-on-a-chip)

4. – Liver ECM engineered 3D scaffolds

5. – Liver ECM bio-printed last generation organoids

Precision-Cut Liver Slices

Olinga P and Schuppan D

Hepatology. 2019 Apr 4. doi: 10.1002/hep.30651. [Epub ahead of print]

Spheroids, Organoids, Lab-on-a-chip

2D 3D

3D Models to Study Liver Pathophysiology

1. – Precision-Cut Liver Slices (PCLS)

2. – Spheroids and Organoids

3. – Microfluidics and Microphysiology Systems (Lab-on-a-chip)

4. – Liver ECM engineered 3D scaffolds

5. – Liver ECM bio-printed last generation organoids

Reproducing human non alcoholic fatty liver under lipotoxic stress

De novo lipogenesis and cholesterol synthesis are increased in the human liver lipotoxic system

3D Models to Study Liver Pathophysiology

1. – Precision-Cut Liver Slices (PCLS)

2. – Spheroids and Organoids

3. – Microfluidics and Microphysiology Systems (Lab-on-a-chip)

4. – Liver ECM engineered 3D scaffolds

5. – Liver ECM bio-printed last generation organoids

3D Human Healthy and Fibrotic Liver ECM

Explanted cirrhotic liver

Donor healthy human liver

Mazza G et al, Nature Scientific Report 2015; Mazza G et al, Nature Scientific Report 2017

3D Scaffold Bio-engineering: Hepatocytes

Mazza G et al. Sci Rep 2017

3D Scaffold Bio-engineering: Stellate Cells

Mazza G et al. Sci Rep 2017

HSC Gene Expression: 2D vs. 3D

Caon E., et al. Unpublished

LX2 cell line Primary hHSC

Primary hHSC Gene Expression: Normal vs Cirrhotic Scaffolds

Caon E., et al. Unpublished

• LTC repopulated with Hep G2 (hepatocyte-like cell line) and LX2 (hepatic stellate cell line) (first as monoculture, then as co-culture)

• Mimic steatosis by treating with Palmitic Acid (PA) or Oleic Acid (OA) every 24 hours for 7 days

• LTC quadruplicates with 100 µM PA/OA or 1% isopropanol (vehicle control)• LTC culture stopped at Day 14 to evaluate extent and distribution of steatosis

A Tissue-engineered 3D Model of NAFLD Employing Human ECM

Targets for Evaluation Method of Analysis

Ultrastructure of ECM and Cell Distribution Histology

Fat Accumulation, Extent of Steatosis Triglyceride LevelsOil Red O Staining (lipid droplet internalisation)

Insulin Sensitivity levels of insulin-stimulated phosphorylation of AKT and Insulin Receptor Substrate-1 (IRS-1)

Production of pro-inflammatory factors Multiplex ELISA

Cell Viability MTS

Cytotoxicity Western BlotTUNEL assay for in situ studies

Cell Death Pathways Western Blot, Immunohistochemistry

Production of lipotoxic mediatorsModulation of key lipid metabolic pathways

QT-PCR

Hepatic Stellate Cell Activation Markers for fibrogenesis, markers of myofibroblastic activation (e.g. cytoskeletal proteins, collagen type I secretion)

Collagen Deposition Sirius Red Staining

NAFLD 3D Model: Experimental Objectives

3D long-term with chronic treatment Vs. 2D short term models

Optimization:1. FBS concentration2. FFAA concentration3. Insulin concentration

HepG2 Vehicle Control HepG2 100mM Oleic Acid HepG2 100mM Palmitic Acid

A Tissue-engineered 3D Model of NAFLD Employing Human ECM

Longato L., et al. Unpublished

A Tissue-engineered 3D Model of NAFLD Employing Human ECM (Relative Gene Expression)

Adipose differentiation-related protein/ perilipin 2/ ADRP/adipophilin (lipid droplet protein)

Stearoyl-CoA desaturase-1 (fatty acids metabolism) Fatty acid synthase (de novo fatty acids synthesis)

Carnitine palmitoyltransferase I (fatty acids oxidation) Sterol regulatory element-binding protein 1 (regulation of fatty acids biosynthesis)

Acetyl-CoA carboxylase (de novo fatty acids synthesis)

Longato L., et al. Unpublished

Does not satisfy the criteria

Does partially satisfy the criteria

Does satisfy the criteria.

3D Human Scaffold Cultures Vs. Other 3D Systems

3D Printed in ECM Bio-Inks

3D Models to Study Liver Pathophysiology

1. – Precision-Cut Liver Slices (PCLS)

2. – Spheroids and Organoids

3. – Microfluidics and Microphysiology Systems (Lab-on-a-chip)

4. – Liver ECM engineered 3D scaffolds

5. – Liver ECM bio-printed last generation organoids

Worm-like micelles Sphere stage

Synthetic diblock copolymer mixed with human ECM solution:

•Human liver ECM solution•2-hydroxypropylmethacrylate (HPMA)•Poly(glycerolmonomethacrylate) (PGMA) macro-CTA

• Thermo-responsive hydrogels (e.g Matrigel) with human tissue-specific ECM (healthy – possibility to develop disease Hep-Gel)

• Cells are mixed with the soluble form and then dispensed into well plates

• Low volume and low cell concentration (1ul/333.3 cells)

• Currently used in 48 well plates (50ul/well) or 96 well plates (10ul/well)

• Spheroids and HTS assays

Hep-Gel: Liver ECM last generation (LG) organoids for HTS*

*HTS: high throughput screening

Bio-printed Cholangiocytes: Ductal Formation

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Hep-Gel: Primary Hepatic Stellate Cells LG Organoids

Caon E., et al. Unpublished

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Caon E., et al. Unpublished

Hep-Gel: Primary Hepatic Stellate Cells LG Organoids

Mazza, et al. Hepatol Commun, 2018

Liver tissue engineering: From implantable tissue to whole organ engineering

“Tissue engineering and regenerative medicine research is currently underfunded, receiving less than $500 million annually in the United States compared to $5 billion for cancer and $2.8 billion for human immunodeficiency virus/acquired immune deficiency syndrome1.”

1 Moses, H, Matheson, DH, Cairns‐Smith, S, George,

BP, Palisch, C, Dorsey, ER. The anatomy of medical

research: US and international comparisons. JAMA

2015; 313: 174‐ 189.

UCL Institute for Liver and Digestive Health: Tissue Engineering Laboratory

UCL ILDHGiuseppe MAZZAKrista ROMBOUTSWalid Al-AkkadLuca FrenguelliLisa LongatoKessarin ThanapiromMaria Giovanna ViliaElisabetta CaonMartina MarraliEric FelliBailin ChenYutaka YasuiZhen Zhen Zhang

Kevin MooreDouglas Thorburn

UCL Div. of SurgeryBrian DavidsonBarry FullerAmir GanderJoerg PollockBettina Hansen

UCL Cellular PathologyAlberto QuagliaTu Vinh LuongAndy Hall

UCL Institute for Child Health, Div. Of SurgeryPaolo De CoppiPanagiotis MaghsoudlouLuca Urbani

Armando Del Rio HernandezBenjamin Robinson

Ludovic VallierFotios Sampaziotis