A 3D in-vitro model of hepatic fibrosis for biomedical applications

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Universit`

a degli Studi di Pisa

DIPARTIMENTO DI BIOLOGIA

Corso di Laurea Magistrale in Biologia molecolare e cellulare

Tesi di laurea magistrale

A 3D in-vitro model of hepatic fibrosis for

biomedical applications

Candidata:

Wendy Balestri

Relatori:

Prof. Arti Ahluwalia Dr. Giorgio Mattei

Correlatori:

Prof. Aldo Paolicchi Dr. Vanna Fierabracci

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Abbreviations

α SMA αsmooth muscle actin

3D Three-dimensional

BSA Bovine Serum Albumin

ECM Extracellular matrix

EGF Epidermal growth factor

ELISA Enzyme-linked immunosorbent assay EMT Epithelial mesenchymal transition

FBS Fetal Bovine Serum

GFP Green fluorescent protein

HCV Hepatitis C Virus

HPCs Hepatic progenitor cells HSCs Hepatic stellate cells

LDH Lactate Dehydrogenase

LSECs Liver sinusoidal endothelial cells MET Mesenchymal-to-epithelial transition

MFs Myofibroblasts

MSC Mesenchymal stem cells

mTG microbial transglutaminase

PBS Phosphate Buffered Saline

PDGF Platelet-derived growth factor

PEG Polyethylene glycol

PFs Portals fibroblasts

PGA Glycolic acid

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Abbreviations

PLGA Lactic glycolic acid

ROS Reactive oxygen species

SDS Sodium dodecyl sulfate

TBS-T Tris-buffered saline Tween TGFβ Transforming growth factor β TNFα Tumour necrosis factorα

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Chapter 1 Abstract

The liver is the primary gland involved in the metabolism of xenobiotics, drugs and toxic and waste substances. It has a high regenerative capacity, due to the proliferation and differentiation of liver cells that restore the organ function. However, when the liver damage is severe and prolonged, due to the onset of chronic liver diseases, the inflammatory response and the repair mechanisms that are activated lead to a progressive fibrosis.

Fibrosis is a dynamic process that involves the progressive accumulation of extracellular matrix in an attempt to repair the damage caused by differ-ent kinds of pathological conditions. Advanced liver fibrosis may result in cirrhosis, liver failure, and portal hypertension and can eventually lead to hepatocellular carcinoma. Fibrosis is promoted by myofibroblasts, which are activated by inflammatory cytokines (such as TGFβ) and may derive from different types of hepatic cells, among which the main are stellate cells. Interestingly, it was demonstrated that the acquisition of fibroblast- and myofibroblasts-phenotypes can arise during the complex biological process known as epithelial-mesenchymal transition (EMT).

The studies focusing on the physiopathological mechanisms associated with liver damage, degeneration and fibrosis are of great interest, also to better identify the risk factors associated with these phenomena. In this perspec-tive, substantial efforts have been made to develop suitable in vitro and in vivo models mimicking liver fibrosis.

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Abstract

For in vitro models, primary cultures of human hepatocytes are the refer-ence model, but the difficult accessibility and the inter-individual variability, turn the choice on immortalized hepatic cell lines. Several in vitro models are based on two-dimensional cultures or co-cultures of different hepatic cell types; however, a more faithful reproduction of the in vivo situation can be obtained with three-dimensional models (3D) of cell cultures. Indeed, the 3D model allow to exploit the interactions of cells with the extracellular matrix, as well as with other cells. In recent years, methods have been developed for 3D culture based on the use of scaffolds of smart materials, of natural (collagen, extracellular matrix decellularized and digested ...) or synthesis origin (polyethylene glycol, PuraMatrixT M_{...), that have the function of an}

extracellular matrix, capable of supporting cell growth and mimicking poros-ity, permeability and mechanical stability of the in vivo conditions.

The aim of this thesis was to set up a 3D model of liver fibrosis by developing a gel matrix that can be stiffened with biocompatible cross-linker and thus used to encapsulate hepatocytes.

Recently, it was shown that the hardening of the extracellular matrix may result in mechanical activation of TGFβ, leading to the activation of the EMT process, causing the expression of mesenchymal markers (vimentin), to the detriment of those epithelial (E-cadherin).

Initially, suitable experimental conditions were optimized to obtain 3D cel-lular cultures of human hepatoma HepG2 encapsulated into collagen gels. Cell viability and proliferation were assessed by Live-Dead test, LDH assay and Trypan blue exclusion assay. The secretion of albumin was selected as a marker of metabolic functionality. Unlike other methods reported in the literature and based on the use of compounds/conditions with unwanted or cytotoxic side effects, the stiffening of our collagen gels was obtained via a fully biocompatible cross-linker, i.e. the microbial enzyme transglutaminase (mTG), that catalyzes the formation of cross links between proteins. The micromechanical properties of gels were analysed by nanoindentation and the effects of gel stiffening on cell morphology, vitality and proliferation were

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Abstract

was evaluated by immunofluorescence and western blot analysis.

Finally, in an effort to set up a more biomimetic model, the preparation of gels with a decellularized and subsequently digested hepatic extracellular matrix obtained from porcine liver was also taken into account.

The results obtained indicate that the 3D collagen model is suitable to study the effects of gel stiffening by mTG up to 96 hours in culture. The stiffening of gels by low cytotoxic mTG activities resulted in a reduced secretion of al-bumin and in an increased expression of vimentin (i.e. two markers of EMT). These results suggest that also small changes in the mechanical properties of extracellular (micro)environment could be in support of a process - the EMT - which is supposed to contribute to hepatic fibrogenesis.

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Chapter 2 Introduction

2.1 The Liver

Liver is an extramural gland that can secrete both exocrine and endocrine material; it is positioned below the diaphragm and located on the right side, between the transverse colon and the stomach. It is the largest gland of the human body and performs several functions:

• It produces bile, needed to emulsify fats and enable their absorption from the intestine;

• As regards the metabolism, gluconeogenesis occurs in this gland, for the production of glucose, in addition to cholesterol and triglycerides synthesis;

• It produces clotting factors such as fibrinogen and thrombin; • It stores up vitamin B12, iron and copper;

• It removes toxic substances from blood (ammonia, toxins, waste...) and molecules that do not serve or work anymore (hemoglobin);

• Until the sixth month of intrauterine life, the liver is the most im-portant hematopoietic organ. In case of splenectomy, hemocatheresis occurs in the liver in order to compensate the lack of spleen.

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2.1. The Liver

The human liver has a remarkable capacity for regeneration. This capac-ity is due to hepatocyte proliferation, that usually are quiescent. Cells need several hours to enter in the cell cycle, to complete G1 fase and to replicate DNA. Hepatocyte proliferation is synchronized and it’s followed by the syn-chronous replication of non-parenchymal cells (Kupffer cells, endothelial cells and stellate cells).Unlike other cell, where proliferation and differentiation are two mutually exclusive events, in liver regeneration, mature hepatocytes en-ter again in the cell cycle and proliferate, thus keeping the vital function of the liver. Excluding the Transforming growth factor β (TGFβ) that is an autocrine factor, replication depends on paracrine stimulation of cytokines, such as HGF and IL-6 and polypeptide growth factors, produced by liver non-parenchymal cells (Kumar et al., 2014).

2.1.1 Liver cells

Hepatocytes are the predominant cell type in the liver parenchyma and are essential, along with the blood vessels, for organization and proper dialogue between the different cell types. Mature hepatocytes, with their localization in liver lobes and paracrine activity, are really crucial for a correct organiza-tion and funcorganiza-tion control of biliary cells and hepatic stellate cells. Endothelial cells, cholangiocytes and stellate cells, on the other hand, are essential to keep a correct hepatocyte function (Cicchini et al., 2015).

Liver sinusoidal endothelial cells: Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells characterized by fenestrations and the lack of a basement membrane (Wisse et al., 1996 and Iwakiri and Grosz-mann, 2007). This vascular endothelium provides more than just a physical barrier for blood circulation. It actively participates in inflammatory reac-tions by several mechanisms, including secretion of cytokines and chemokines to recruit and activate leukocytes. They have a strong scavenging capac-ity, which mediates the uptake of several waste macromolecules such as hyaluronic acid, collagen α-chains and modified low-density lipoproteins (Li et al., 2011, McCourt et al., 1999 and Malovic et al., 2007).

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2.1. The Liver

Kupffer cells: Kupffer cells also known as stellate macrophages and Kupffer-Browicz cells, are specialized macrophages located in the liver, lin-ing the walls of the sinusoids that form part of the mononuclear phagocyte system. Kupffer cell activation is responsible for early ethanol-induced liver injury, common in chronic alcoholics. After their activation, Kupffer cells synthesize and secrete the pro-inflammatory cytokines tumour necrosis fac-tor α (TNFα) and IL-1β. Both cytokines can potentially cause hepatocyte killing by activation of signal transduction pathways that lead to apoptosis. Furthermore, Kupffer cells secreted cytokines may attract and activate im-mune cells which in certain cases can exacerbate the initial damage (Roberts et al., 2007).

Biliary epithelial cells: Biliary epithelial cells (i.e. cholangiocytes) line the tubular conduits which constitute the biliary tract. In the healthy liver, cholangiocytes contribute to bile secretion via net release of bicarbonate and water. Several hormones and locally acting mediators are known to con-tribute to cholangiocyte fluid/electrolyte secretion. These include secretin, acetylcholine, ATP, and bombesin. Cholangiocytes act through bile-acid in-dependent bile flow, which is driven by the active transport of electrolytes. In contrast, hepatocytes secrete bile through bile-acid dependent bile flow, which is coupled to canalicular secretion of bile acids via ATP-driven trans-porters. This results in passive transcellular and paracellular secretion of fluid and electrolytes through an osmotic effect. Bile acids have been shown to regulate diverse cholangiocyte functions.

Hepatic stellate cells: Hepatic stellate cells (HSCs) reside within the space of Disse in the liver which is formed between the parenchyma (hep-atocytes) and sinusoidal endothelial cells. Under normal conditions, these cells constitute a major storage site for retinoid (vitamin A) in the body. Their role in pathological conditions will be discussed in the next section, considering that they have an important role in liver fibrosis (Godoy et al.,

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2.1. The Liver

2.1.2 Extracellular matrix

A really important role in regeneration and repair is played by extracellular matrix (ECM). The remodelling of the matrix is dynamic and is an integral part of morphogenesis, regeneration, wound healing, chronic fibrosis, tumours invasiveness and metastasis. Its functions include:

• To provide mechanical support for anchorage and migration of cells and maintain cell polarity;

• To modulate the proliferation, through integrins receptors;

• To keep cell differentiation, a process largely mediated by integrins; • To serve as a scaffold for regeneration. The ECM is essential to

main-tain the normal tissue architecture and its integrity is required to sup-port the regenerative process. On the contrary, the reparative process may take place thus producing the deposition of collagen by fibroblasts and the formation of a scar.;

• To create tissue microenvironments;

• To accumulate and submit regulatory molecules to target cells. Some growth factors are stored in the matrix of certain tissues and may be thus released rapidly as a result of injury.

ECM is made by three types of molecules: fibrous structural proteins, such as collagens and elastins, that gives resistance to the matrix and elas-ticity to tissues; adhesion glycoproteins, that combine the elements of the ECM each other and to cells; and proteoglycans and hyaluronic acid that give to the matrix a mechanical resistance and have a lubricating effect.

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2.1. The Liver

Among the components of ECM, collagen is the main structural protein in connective tissues and the most abundant protein in the animal kingdom. Moreover, collagen has been extensively employed for the preparation of in vitro models of ECM and it has also many medical uses.

It is characterized by a repetitive amino acid sequence, in which in the third position is always present a glycine and it contains the specialized amino acids 4-hydroxyproline and hydroxylysine. So far, several types of collagen have been identified and described (Table 2.1). Fibrillar collagens (I, II, III, V and VI type) are made by long uninterrupted triple helix sequences. Type IV collagens are constituted by long triple helix sequences, but they are discontinuous and form β-sheets instead of fibrils. For each type of colla-gen three specific chains are assembled to form a triple helix (pro-collacolla-gen). Procollagen is secreted and proteases remove terminal fragments to form the basal units of the fibrils. Then, crusaders bonds are formed between adjacent fibrils that stabilize the fiber and are essential to ensure the strength of the collagen supply.

Type Characteristics Distribution

I Bundles of banded fibers with high tensile strength

Skin (80%), bone (90%), tendons, most other organs

II Thin fibrils; structural proteins Cartilage (50%), vitreous humor III Thin fibrils, pliable Blood vessels, uterus, skin (10%)

IV Amorphous All basement membranes

V Amorphous/fine fibrils 2-5% of intestinal tissues, blood vessels

VI Amorphous/fine fibrils Intestinal tissues

VII Anchoring filament Dermal-epidermal junction

VIII Probably amorphous Endothelium-Descemet membrane

IX Possible role in maturation of car-tilage

Cartilage

Table 2.1: Types of collagen. Adapted from Robbins, Pathologic basis of disease.

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2.2. Liver Fibrosis

such as pore structure, permeability, hydrophilicity and it is stable in vivo (S. M. Oliveira et al., 2010).

2.1.3 Wound repair and scar forming

In severe or chronic pathological processes affecting both parenchymal cells and stromal framework, tissue destruction may occur and repair cannot be accomplished solely by regeneration of parenchyma. Attempts to repair tis-sue damage then occur by replacement of parenchyma by connective tistis-sue, i.e. by deposition of collagen and other matrix components which finally may produce fibrosis and scarring (Robbins, Pathologic basis of disease) The processes necessary for scar formation are:

1. Inflammation

2. Angiogenesis, i.e. the formation of new blood vessels

3. Fibroblast migration and regeneration

4. Deposition of ECM

5. Remodelling of connective tissue, i.e. the maturation and organization of the fibrous tissue

The inflammatory process triggered by injuries removes the damaged tis-sue components, promote angiogenesis and eventually stimulate the deposi-tion of ECM. If the pathological stimulus persists, the inflammadeposi-tion becomes chronic and cause the accumulation of connective tissue, i.e. fibrosis.

2.2 Liver Fibrosis

The etiopathogenic mechanism of fibrosis of chronic inflammatory process produced by diseases is similar to scarring but, in this case, the inflamma-tory stimulus (infections, autoimmune reactions, traumas and diseases) that activates the ordered chain of events is persistent and causes organ damage, which often leads to a definitive functional impairment.

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2.2. Liver Fibrosis

The continuity of the disease process causes chronic inflammation, followed by the proliferation and activation of macrophages and lymphocytes and production of fibrogenic growth factors and inflammatory cytokines. Acti-vated macrophages produce TGFβ, an important fibrogenic factor involved in chronic inflammatory diseases. This factor stimulates the migration and proliferation of fibroblasts and the synthesis of collagen and fibronectin, with the intent to limit the damage. Moreover, it inhibits the metalloprotease. TGFβ is released also in the tissues as a result of cell death and production of free radicals. In the liver, the main cells that are stimulated by TGFβ are stellate cells (Kumar et al., 2014).

Healthy Liver Fibrotic Liver Cirrhotic Liver

Liver Transplant Liver Cancer Chronic Injury

Viral infection Autoimmune injury Toxic metabolites and drugs Regular and excessive consumption of alcohol Hereditary defects

Genetic polymorphism Epigenetic marks Cofactors (obesity, alcohol…)

Liver failure Portal hypertension Inflammatory damage Matrix deposition Apoptosis Angiogenesis Disrupted architecture Loss of fuction Aberrant hepatocyte regeneration

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2.2. Liver Fibrosis

Chronic liver diseases are characterized by lasting damage to hepatocytes induced by:

1. Chronic infections with hepatotropic viruses (mainly hepatitis B and C viruses);

2. Autoimmune injuries;

3. Toxic metabolites and drugs;

4. Regular and excessive consumption of alcohol;

5. Hereditary defects.

As shown in figure 2.1, persistent fibrogenesis is responsible for the pro-gression of any form of chronic liver disease to the end-points of liver cirrhosis and hepatic failure.

Cirrhosis is currently defined as an advanced stage of fibrosis and is character-ized by the formation of regenerative nodules of the parenchyma surrounded and separated by fibrotic septa. The development of cirrhosis is associated with significant changes in organ vascular architecture, development of por-tal hypertension and related complications.

Fibrosis can proceed through four distinct patterns of fibrosis related to the underlying etiology:

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2.2. Liver Fibrosis

Pattern of fibrosis Aetiology and mechanism Cell Type

Bridging fibrosis

Chronic viral infections Autoimmune diseases

Chronical activation of wound healing Oxidative Stress HSC/MFs Portal MFs Bone marrow-derived MFs Perisinusoidal fibrosis

Non-alcoholic fatty liver dis-ease (NAFLD) to non- alco-holic steatohepatitis (NASH) Alcoholic hepatitis (ASH) Oxidative Stress

HSC/MFs

Biliary fibrosis

Chronic cholestatic cholan-giopaties

Derangement of interaction between cholangiocytes and mesenchymal cells

Cholangiocytes transition into MFs (EMT)

Oxidative Stress

Portal MFs

EMT-derived MFs

Centrilobular fibrosis Not correlated with chronic liver disease

Table 2.2: Patterns of liver fibrosis. HSC, hepatic stellate cells; MFs, myofibroblasts;

EMT, epithelial-mesenchymal transition.

2.2.1 Hepatic myofibroblasts

Irrespective of the specific etiology or the prevalent pattern of fibrosis, liver fibrogenesis is sustained by hepatic myofibroblasts (MFs). They constitute a heterogeneous population of cells, mostly positive for αsmooth muscle actin (αSMA), which are mainly found in fibrotic and cirrhotic livers. Hepatic MFs are highly proliferative and contractile cells which actively contribute to chronic liver disease progression by means of their multiple phenotypic responses, including:

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2.2. Liver Fibrosis

• Excess deposition of extracellular matrix components and its altered remodelling;

• Synthesis and release (paracrine/autocrine) of several critical growth factors able to sustain and perpetuate not only fibrogenesis but also chronic inflammatory responses and neo-angiogenesis.

Hepatic MFs represent a unique and critical cellular crossroad able to inte-grate incoming paracrine and autocrine signals (Forbes and Parola, 2011).

There are at least four mechanism that can induce fibrosis in which MFs play a key role:

• Chronical activation of inflammatory response (which predominate in viral infections and autoimmune responses);

• Oxidative Stress (mainly associated to metabolic or alcoholic etiology); • Derangement of epithelial-mesenchymal interactions (chronic

cholan-giopathies);

• Epithelial-mesenchymal transition (EMT).

2.2.2 Hepatic stellate cells

Under conditions of chronic liver injury, quiescent HSCs have been reported to undergo a process of activation which involves significant changes in mor-phology and phenotypical responses observed in either human or rat HSC when cultured on plastic substrate (Friedman, 2008). For the activation, there is an early response stimulated by paracrine signals, lead to a transient and reversible, contractile and pro-fibrogenic cellular phenotype, character-ized by a rapid induction of platelet-derived growth factor (PDGF) receptor expression.

HSC/MFs are characterized by the following properties:

• High proliferative capacity: promoted by mitogens such as PDGF, bFGF, angiotensin, VEGF and others;

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2.2. Liver Fibrosis

• Migration and chemotaxis capacity: essential to reach the site of injury, this property is induced stimulated by PDGF, VEGF and angiotensin; • ECM synthesis and remodelling capacity: fibrogenesis progression is characterized by the replacement of low-density basement membrane of the subendothelial space of Disse with fibril-forming matrix, a scenario that is believed to result primarily from a disequilibrium between excess deposition of fibrillar collagens as well as of other ECM components and a reduced/altered degradation and remodelling of fibrotic ECM. Collagen production is induced by TGFβ, produced by Kupffer cells and subsequently by HSC itself. HSC/MFs also produce metal protease inhibitors;

• Contractility capacity: HSC/MFs show features of smooth muscle cells and contractility; by responding to vasoactive mediators, they con-tribute to increased portal resistance during early stages of fibrosis. HSC/MFs are also able to sense matrix stiffness and respond to this by upregulating their pro-fibrogenic phenotype;

• Pro-inflammatory response features: HSC/MFs release a number of pro-inflammatory molecules, chemoattractants and chemokines. More-over, HSC/MFs also behave as target cells for inflammatory cytokines and other pro-inflammatory signals, including reactive oxygen species (ROS), generated as a consequence of hepatocyte injury and necrosis, apoptotic bodies, bacterial endotoxin or other endogenous activators; • Role in angiogenesis: by producing VEGF;

• Immune-modulatory roles: HSC/MFs can control leukocytes behaviour and they can suppress lymphocytes (local immunotolerance);

• Role in regeneration and cancer: producing growth factors for either mature epithelial cells or hepatic progenitor cells (HPCs), bipotent pro-genitors of hepatocytes and cholangiocytes. However, HSC/MFs have been shown to express also the stem cell marker CD133. This has led to

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2.2. Liver Fibrosis

the controversial hypothesis that HSC/MFs may directly differentiate into epithelial stem or precursor cells.

2.2.3 Portal fibroblasts

Portals fibroblasts (PFs) can differentiate into MF in case of chronic damage conditions. Their main role is in biliary fibrosis, since the damage to the bile duct cells is a prerequisite for differentiation of PFs in MFs. These cells, once damaged, express TGFβ and release growth factors and pro-inflammatory mediators. These factors are responsible for the onset of differentiation in MFs.

2.2.4 Bone-marrow-derived fibrotic cells

They can contribute to the regeneration and fibrosis of solid organs. They transdifferentiate within the adult tissue to form epithelial cells. It seems that it is the mesenchymal stem cells (MSC) that transform hematopoietic stem cells in HSCs (Forbes and Parola, 2011).

2.2.5 Fibrogenic cells from Epithelial-Mesenchymal

Tran-sition

The EMT is a complex phenomenon by which several types of epithelial polarized cells lose cell-cell connections and acquire mesenchymal character-istics of motility and invasiveness.

Hallmarks for EMT include increased expression of vimentin, nuclear local-ization of β-catenin and production of transcription factors able to inhibit E-cadherin expression.

Master EMT inducers have been identified in the transcriptional repressors belonging to the Snail family, Snail (Snai1) and Slug (Snai2); these factors are able to determine EMT induction targeting many epithelial genes start-ing from the direct inhibition of E-cadherin gene transcription.

Many signalling pathways are implicated in EMT induction, including TGFβ, epidermal growth factor (EGF), Wnt, sonic hedgehog (Hh) and Notch, and

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2.3. Animal models for liver fibrosis

activation of proto-oncogenes, such as Src or Ras activation. In particu-lar, TGFβ is considered the master EMT inducer for malignant and non-malignant epithelial cells, including hepatocytes.

When TGFβ binds to its receptors, Smad 2/3 are phosphorylated. Their phosphorylation promotes the recruitment of Smad4, which translocates to the nucleus, it binds Smad3 and together they prevent the expression of genes encoding epithelial markers.

The EMT reverse event, the mesenchymal-to-epithelial transition (MET), allows the mesenchymal cells to redifferentiate into epithelial structures. MET occurs both in physiological (i.e. in ontogenesis) and in pathological situations (i.e. cancer metastatization), where the migrating mesenchymal-like cells that have reached secondary sites reacquire cell-cell contacts and polarity.

In the liver, many cells that undergo EMT have been identified. In par-ticular hepatocytes, when treated with TGFβ, reduce levels of epithelial and liver markers (E-cadherin and albumin) in favour of mesenchymal genes (vi-mentin and αSMA) and acquire motility and invasiveness. (Cicchini et al., 2015)

It has been shown that the activation of TGFβ can also take place me-chanically as a result of the stiffening of the extracellular matrix, i.e. in conditions able to affect cell contractility (Nasrollahi and Pathak, 2015).

2.3 Animal models for liver fibrosis

At present, there aren’t models that represent the totality of liver fibrosis synthoms and mechanisms. There are lots of animals (e.g. rabbit, dog, ape...), but the most used and better known is the rodent. The use of animal model has several advantages:

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• The possibility to have many samples, at different times and perform sequential studies;

• There is a short illness development;

• It’s possible to control and reduce some sources of variability that in human is impossible to follow;

• It’s possible to study genes and pathways with transgenic animals.

Figure 2.2: Animal models for liver fibrosis.

Unlike in vitro models, animals allow to study the whole organ, keep-ing safe the interactions between organ and the rest of the body, includkeep-ing immune and vascular system. Furthermore, you can analyze cell-cell interac-tions, cell-matrix interactions and their variations due to metabolic action or endocrine activities. However, animals aren’t humans, so they don’t develop the same kind of diseases. Mice, for example, can’t be infected by Hepatitis C Virus (HCV) and it is difficult to induce alcohol-dependent diseases, since alcohol is metabolized very quickly.

There are various models using completely different approaches to induce the genesis of liver fibrosis:

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1. Immunologically mediated fibrosis:

• Schistosomiasis haematobium infection • Repetitive Concanavalin A injection

2. Cholestatic fibrosis induced by bile duct ligation

3. Chemically induced fibrosis • Carbon tetrachloride (CCl4)

• Thioacetamide (TAA)

• Dimethylnitrosamine (DMN) 4. Transgenic animals

2.3.1 Immunologically mediated fibrosis

There is the activation of different cells of the immune system like Kupf-fer cells, lymphocytes, monocytes and thrombocytes, which activate hepatic stellate cells. This mechanism can be modulated by Schistosomiasis haema-tobium infection or repetitive Concanavalin A injection, both dependent on upregulation of TGFβ. However, with these approaches only a reduced num-ber of sick animals can be obtained and they can also produce a rupture of the bile ducts rather than a fibrosis.

Schistosomiasis haematobium infection: This parasite causes hep-atic fibrosis in humans. Through the skin penetration, the mature cercariae migrate to the liver and mesenteric veins, where they lay eggs. Some eggs migrate in bowel lumen and are excreted, while the others remain in the liver and induce a strong cytokine response, that induces the formation of eosinophil-rich granulomas. Then, HSCs are activated leading to severe liver fibrosis, portal obstruction and liver malfunction. In mice fibrosis is induced by intravenous injection of the eggs. The consequence is an overexpression of markers of fibrosis, such as αSMA and collagen I and the production of

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higher levels of TGFβ and other cytokines.

Repetitive Concanavalin A injection: Concanavalin A is an activa-tor of T-cells in liver. Multiple injections of this compound can lead to an aggressive immune response, due to an increase of INFγ and TNFα concen-tration, with TGFβ upregulation, which eventually actives HSCs.

2.3.2 Cholestatic fibrosis

Cholestatic fibrosis may occur in humans with primary sclerosing cholangi-tis, but also following bile duct obstruction due to various reasons. Even though a surgical skill is required to have bile duct obstruction in mice, this method produces the activation of HSCs and the consequent fibrosis without any carcinogenic effect. In mice, the gallbladder must be removed, while in rats it is not present.

Bile duct ligation: the interruption of bile flow induces a strong prolif-eration of the duct cells, portal inflammation and rapid portal fibrosis. Bile duct-ligated animals present marked fibrosis after 12 days and liver cirrhosis is present in almost all animals within 4-8 weeks.

2.3.3 Chemically induced fibrosis

Different chemical agents, most of them known carcinogens, are used to mimic alcoholic and non-alcoholic steatohepatitis as well as fibrosis and cirrhosis. The most common chemical compounds used to induce liver fibrosis are:

Carbon tetrachloride: It is a hepatotoxin that causes acute hepatic damage and, when given repetitively at a low dose, induces liver fibrosis. A dose of 0.5-2 ml/kg body weight, administrated twice per week, is enough to induce fibrosis in few weeks. In liver, trichloromethyl radicals (CCl3)

are formed, and this worsens hepatocytes damage, due to radicals and lipid peroxidation reactions that contribute to the activation of stellate cells, even-tually leading the production of extracellular matrix. Like the other models,

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TGFβ is upregulated and maintained throughout the fibrotic process. Ei-ther mice and rats can be used, but mice are preferred as they metabolize tetrachloride better.

Thioacetamide: It is a selective hematotoxin well known to cause liver fibrosis as well as tumours such as cholangiocarcinomas. Its metabolic prod-ucts are highly reactive compounds that cause oxidative stress. It is admin-istered orally, diluted to 0.03% in water. This method has few side effects and is highly reproducible. To increase the levels of fibrosis, it is applied with ethanol.

Dimethylnitrosamine: It is a carcinogenic chemical that is typically injected intraperitoneally three times a week for six consecutive weeks. It is widely used in studies on cancer, but can also cause distortions of lobular architecture and liver cirrhosis with portal hypertension and complications like loss of liver function. It causes reliable induction of liver fibrosis and it has been shown to induce fatty degeneration of hepatocytes, activation and proliferation of hepatic stellate cells, activation of Kupffer cells and secretion of TGFβ.

2.3.4 Transgenic animals

Transgenic mice offer the opportunity to study in deep other pathways and mechanisms involved in liver fibrosis.

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TGFβ : Considering that TGFβ administration causes liver fibrosis and that in all the previously described models there is an overexpression of such cytokine, mice with TGFβ gene modified was created. Different approaches and methods were used to obtain transgenic mice: In a study by Kanzler et al. (Kanzler et al., 2001), the porcine TGFβ gene was placed under the con-trol of the albumin promoter/enhancer elements; secretion of mature porcine TGFβ by mouse hepatocytes lead to hepatic fibrosis in vivo and increased the mitotic activity of hepatocytes. In a study by Clouthier et al. (Clouthier, Comerford, and Hammer, 1997), TGFβ gene was placed under the control of the rat promoter/enhancer elements of phosphoenol pyruvate carboxykinase, that allows a constitutive TGFβ expression in liver, kidney and adipose tis-sue. This promoter can be controlled and it’s possible to have different levels of fibrosis by TGFβ concentration modulation. Finally, in another study, porcine TGFβ gene was placed under the control tetracycline- dependent promoter, in order to control TGFβ expression. It seems that silencing the promoter, fibrosis can regress (Weiler-Normann, Herkel, and Lohse, 2007).

Other transgenic mice were created by modifying PDGF (Pinzani et al., 1994) and TIMP-1 genes (Yoshiji et al., 2000).

During the past few decades, the public consciousness regarding animal welfare, especially in Europe, has dramatically changed. As a consequence of the debates surrounding this issue, in 2013 all the EU member states have incorporated into their national legislation the criteria of the EU Directive 2010/63 on the protection of animals used for scientific purposes. This di-rective contains 66 articles and lays down rules for protection of nonhuman primates, animals taken from the wild, stray and feral animals of domestic species and animals bred for use for invasive or non-invasive animal experi-mentation or other scientific purposes (i.e. the so-called ”procedures”). This directive promotes the use of the principle of the 3Rs of Russell and Burch (Replace the animal model, Reduce the number of animals necessary, Refine: “simply to reduce to an absolute minimum the amount of stress imposed on those animals that are still used”). Practically, all the member states must

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2.4. In vitro models for liver fibrosis

ensure that, when it’s possible, a scientifically satisfactory method or exam-ination strategy that does not involve the use of animals is used (Liedtke et al., 2013). In Italy, the Directive has been complemented by Law 26 of 2014.

2.4 In vitro

models for liver fibrosis

In vitro analysis allow to study single phenomena, isolating them from the rest, so it can be clearly distinguished. As for the hepatic fibrosis, the liter-ature is rich in studies on hepatic stellate cells, the main players involved in fibrotic processes. The easiest way to study liver fibrosis in vitro is through monoculture of cells. Next up a detailed description of the most commonly used cellular models.

2.4.1 Hepatic stellate cells

Primary hepatic stellate cells : Primary HSCs, taken directly from a healthy hepatic tissue, well represent the in vivo conditions. However, this model results complicated due to cells isolation and cultivation. Isolation is made by centrifuge on a density gradient. HSCs have a low density because of their abundant lipid content. However, this method can’t be used with young animals or ill livers because of their lack of lipid content or poor purity. One solution could be to make a sorting selecting cells that have vitamin A excited by ultraviolet laser. The problem is that this process is slow and complicate, and, like the other primary cultures, the life length of the cells is limited. Moreover, despite the improved techniques of isolation and a high purity, the cultures may be contaminated with other liver cells. Finally, the creation of human HSCs cultures is limited by a general lack of human biological material for research purposes.

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Immortalized hepatic cell lines

Immortalized cell lines that can be used as in vitro model and they can be isolated from mouse, rat and man.

Mice hepatic stellate cells: One of the first lines obtained was GRX, where the cells were taken from a fibrotic liver granuloma from mice infected with Shistosoma mansoni. Cultured GRX show a myofibroblastic pheno-type and grow in the hills and valleys due to low contact inhibition. When they are transferred to a culture medium containing insulin and retinol, they adopt a phenotype that stores fat and are organized in a regular monolayer. Both phenotypes express collagen type I, III and IV, fibronectin, laminin and αSMA, although in the second phenotype occurs at a lesser extent. (Boro-jevic et al., 1985)

The A640 IS-HSCs were isolated from male imprinting control region in mice that were transduced with the large T antigen of SV40 virus. This cell line is sensitive to temperature, i.e. assume a myofibroblastic pheno-type and proliferative phenopheno-type at temperatures lower than 33◦_{C, whereas}

a morphology more similar to that of HSCs can be obtained when cultured at 39◦_{C. Both phenotypes express collagen type I, III and IV, fibronectin,}

laminin and αSMA (Kitamura et al., 1997).

A recently produced cell line is the Col-GFP, i.e. HSCs isolated from transgenic mice treated with CCl4 for 8 weeks, that express green fluorescent

protein (GFP) under the control of the promoter of the gene for collagen I. To immortalizing these cells was used a lentiviral vector containing SV40 large T antigen and the resistance to hygromycin. The resulting cells were characterized by the expression of collagen I and IV, fibronectin and αSMA (Meurer et al., 2013).

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Human hepatic stellate cells: The LI90 is the first human HSCs im-mortalized line. It was originated from an epithelioid hemangioendothelioma localized in the right lobe of a 55 year old Japanese woman, through cholecys-tectomy. These cells have a polygonal shape and a high rate of proliferation and their massive growth is ensured by a lack of contact inhibition. They produce collagen type I, III, IV and V, fibronectin, and laminin and αSMA (Meurer et al., 2013).

The hTERT-HSCs have been produced to prevent the aging of HSCs in culture. The HSCs has been taken from a healthy liver and hTERT has been inserted under the control of a constitutive cytomegalovirus promoter. The most popular are the LX-1 and LX-2 generated by transduction with TSV40. Both cell lines express collagen I and IV, fibronectin, vimentin, αSMA and TGFβ receptors. LX-2 is also able to produce metalloproteases (Schnabl et al., 2002).

Co-cultures: With the monoculture it is not possible to study the im-portant interactions that are created between the various cell types that can be found within the liver. The simplest thing to do is to isolate the sick cell lines and then plate them together in a single layer. By varying the seeding density of the different cell types, it is possible to modulate the degree of interaction between the cells. To study the role of soluble factors it is pos-sible to plate the cells into a transwell system in which a couple of cell lines can grow separately. Transwell permeable supports also allow to study the interactions between two different cell types that are mediated by secreted factors (Yanguas et al., 2016).

2.4.2 Precision-cut liver slices

Precision-cut liver slices are an excellent model that allows to study fibrotic processes in a multicellular system in which cell-cell and cell-matrix interac-tions are preserved. Slices of 5-8 mm in diameter with a thickness ranging

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from 175 and 250 µm must be obtained in order to allow oxygen and nutrients diffusion into inner cells. The thickness must be sufficient to maintain a more favourable possible ratio between cells damaged at the edges and the mass of living cells. There are two main groups of culture systems of precision-cut liver slices, which can be divided into (Olinga and Groothuis, 2001):

• Continuously submerged culture systems: the slices are floating within the culture medium while the system is gently shaken, or the slices are placed on a stainless-steel grid while the culture medium is magnetically stirred.

• Dynamic organ culture systems: the slices are alternately exposed to the gas phase and the culture medium by placing the slices on inserts in a container, which is rolled or rocked during incubation.

In any case, the slices are incubated at 37◦_{C with 5% CO}

2 and, for

long periods of incubation, the culture medium is necessary. The maximum duration of incubation is not yet clear, however incubations of slices for 48 hours were described. It has been seen that for long periods it is best to use a crop of dynamic type with a rich medium (Van de Bovenkamp et al., 2007).

2.4.3 Parenchymal cells

It is interesting to evaluate the effects of fibrosis on parenchymal cells. Primary cultures of human hepatocytes are considered the reference model, although aspects related to the poor availability of primary hepatocytes and to the inter-individual variability limit their use. For these reasons, it is preferable to use immortalized cell lines of hepatocytes.

The most commonly cell line used for this purpose is represented by hu-man hepatoma HepG2 cells. The cell line HepG2 is a good model for the in vitro study of liver specific functions.

These cells retain certain properties of human liver, such as the ability to synthesize and secrete bile acids and form polarized structures, identified as bile canaliculi (Chiu et al., 1990).

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2.5. 3D models for liver fibrosis

HepG2 cells were also used for the preparation of cellular spheroids, which will be discussed in the next section.

The HepaRG cells are derived from a line of ancestors of human liver cells, which retain many characteristics of human primary hepatocytes, including morphology and expression of key metabolic enzymes, nuclear receptors and drug carriers (Berger et al., 2016). Finally, also the HuH7 cell line, derived from a human hepatocellular carcinoma was used as a suitable in vitro model (El-Shamy et al., 2015).

As for the hepatic stellate cells, also the hepatocytes can also be placed in co-culture. It has been seen that immortalized cell lines of hepatocytes cultured with human stem cells derived from adipose tissue, human umbilical vein endothelial cells, fibroblasts, and other non-parenchymal cells, maintain cellular functions of all cell types and biotransformation ability of hepatocytes (Bachmann et al., 2015).

2.5 3D models for liver fibrosis

Most systems for the cultivation of hepatocytes are 2D monolayer cultures, which are based on the coating of surfaces with purified components of the extracellular matrix such as collagen. The cultivation of hepatocytes under monolayer conditions has been extensively used for drug-testing, as well as for investigating protective drug effects and underlying mechanisms of drug toxi-city. Unfortunately, under these conditions, hepatocytes de-differentiate and lose their biotransformation capacity within a few days, conferring human hepatocytes a very limited time-frame for toxicological studies (Bachmann et al., 2015). To reproduce the three-dimensional characteristics of the liver is important because the in vivo environment influences the behaviour of cells and their function through a variety of insoluble and soluble signalling fac-tors, in addition to provide mechanical, chemical and physical signals. A 3D scaffold should provide an adhesive substrate that mimics the extracellular

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In addition, permeability is required to maintain the metabolic activities, carry large macromolecules and allow cell-cell interactions.

2.5.1 Hydrogels

Hydrogels present water-swollen polymers derived from natural or synthetic resources. Hydrogels derived from natural resources are made by ECM and other proteins. Among the most used there are those made by proteins, such as collagen and Matrigel®, but also polysaccharides, such as chitosan, dextran and alginate. The collagen is extracted, usually, by rat tail ten-dons; Matrigel®is a mixture of matrix proteins secreted by mouse sarcoma Engelbreth-Holm-Swarm cells. Hydrogels derived from natural substances are biocompatible and biodegradable and they promote cellular activities. However, they may contain pathogenic biological or stimulate the inflam-matory response. In addition, there is high variability between batches, so hampers the reproducibility of results. Moreover, the physical properties of such models allow only a poor transport of oxygen and nutrients.

To overcome these problems, synthetic polymers have been created. These are formed by vinyl acetate monomers, acrylamide, lactic acid and, among the most used, the polyethylene glycol (PEG). The latter presents hydroxyl groups that can be easily modified by functional groups, attached to other molecules or bioactive agents. Another self-assembling peptide commercially available is PuraMatrixT M _{(3D Matrix Inc, Waltham, MA, USA). Gelatin is}

often used, but is necessary to create chemical cross link at room tempera-ture to form a stable gelatin. The synthesis of these polymers can be easily controlled and customized, to have a greater range of properties. In addi-tion, the use of polymers reduces the risk of contamination by pathogens, and the stimulation of the immune response. The main disadvantages of these hydrogels are the high costs of adaptation to high-throughput analysis and the low number of available data on hepatocytes. Moreover, they are little biodegradable, have absent intrinsic bioactive properties and, finally, may contain toxic substances (Paleos, 2012).

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Hydrogels are used to seed the cells on one matrix layer. Recently, these substances were used to create a 3D environment in which cells are seeded in the sandwich, i.e. between two different layers of gel, or are included inside the same matrix (scaffold) where they can proliferate, migrate and differen-tiate.

2.5.2 Scaffolds

There are two kinds of methods for producing 3D scaffolds. One is the con-ventional scaffold method that includes fiber bonding, phase separation, par-ticulate leaching, melt molding, gas foaming and freeze drying. Among the conventional manufacturing techniques, the lyophilization of derived natural polymers has been applied for the engineering of tissues of the liver, due to the porosity and the average size of the pores in the 3D scaffolds are controlled by the cooling rate. However, the conventional techniques have several limi-tations, such as being manual-based processes, inconsistent procedures with shape limitations and the use of toxic organic solvents and porogens.

The other types of methods are based on 3D printing scaffolds (Godoy et al., 2013).

Scaffolds, as previously described, can be derived from natural or artifi-cial materials. As regards natural materials, the decellularized extracellular matrix derived from the liver has been used for 3D cultures of hepatocytes because it is resorbable, easy to handle and maintain the liver functions for a long time. Moreover, it provides the 3D organ architecture and the components of the ECM. To get the decellularized matrix there are various methods.

The first used was the whole organ perfusion, by sending detergents through the blood vessels. In addition to perfusion (Uygun et al., 2010), it’s possible to apply a pressure gradient (Prasertsung et al., 2008), immersion and agi-tation. Many methods involve washing and saponification to remove blood

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and debris and to hydrolyze the plasma membrane (Mattei, Di Patria, et al., 2014).

As the natural scaffolds, they are biocompatible, but there are problems concerning the composition of these, which are not always fully known, there may be variability between batches, their preparation is laborious and re-quires a lot of attention.

The artificial scaffolds may be a viable alternative, as they are more eas-ily controllable and less laborious to obtain, although they are often not biocompatible and can be toxic. The materials that may be used are lactic acid (PLA), glycolic acid (PGA) and lactic glycolic acid (PLGA).

With the wide range of natural and synthetic materials available, the challenge for biotechnology companies wishing to develop technologies for the 3D hepatocyte culture routine is therefore the choice of the most suitable material, method of manufacture and final format. Technologies based on natural materials are not ideal for routine use because of the problems with consistency and stability. On the other hand, until recently, technological advances based on synthetic materials have faced difficulties in producing inert, highly porous and reproducible structures that can be presented in an easy-to-use manner. Recently, a porous synthetic scaffold, Alvetex®, has been marketed for routine cell culture of a variety of cell types, including hepatocytes (Bokhari et al., 2007).

2.5.3 Spheroids

The spheroids are multicellular aggregates that, in contrast to monolayer cultures, possess a three-dimensional structure with cells organized in a dif-ferent functional state, i.e. proliferating cells in the periphery and quiescent cells at the center. In the spheroids, liver cells can survive and maintain their specific functions of organs significantly longer compared to monolay-ers, representing therefore a good in vitro model for the simulation of in vivo

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conditions (Casta˜neda and Kinne, 2000).

Figure 2.3: Liver spheroid. Adapted from HepG2 Liver Microtissues 2017.

Spheroids can be created with various methods, such as spontaneous self-assembly in non-adhesive wells/dishes under static conditions, with agitation (rotary culture, rocked culture, Bioreactor), microcavities or in a hanging drop. The most critical point is the size of the spheroid, as spheroids larger than 200-300 microns are at risk to develop necrotic areas, because of the limitation of the oxygen diffusion.

Modulation of extracellular matrix stiffness

In in vitro studies with 3D cultures, it can be interesting to assess how the composition and texture of the extracellular matrix influences cells activity.

To obtain a hardening of the matrix one of the most effective way is to create cross links of the proteins that constitute the matrix itself. There are various methods to create cross links that can be physical, for example with UV light, in which the interactions are ionic and/or hydrophobic, or crys-tallizations. This type of cross-linking has the advantage of being reversible and there isn’t the risk of having potentially harmful chemical reactions for the integrity of the incorporated bioactive agents or cells. However, in vitro stability may be affected by physical interactions, both physiological and me-chanical.

The chemical cross links allow the formation of gel with controllable me-chanical strength and greater physiological stability. These crosslinks can

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be formed by radical polymerization, chemical reactions between comple-mentary groups or by using high-energy radiation. Chemical agents such as formaldehyde, glutaraldehyde, epoxy compounds, and genipin can be used to join the amino-groups of lysines, hydroxylysine, or arginine residues of dif-ferent polypeptide chains with monomeric or oligomeric cross links. Amide cross links may be formed by activation of the carboxylic acid of glutamate or aspartate residues with carbodiimide, followed by reaction of these activated carboxy groups with the amino groups of another polypeptide chain. These chemical modifications, however, reduce the biocompatibility of the material. Cytotoxicity may derive from residues left by crosslinking substances or by the degradation products of the latter (Sung et al., 1999).

An alternative to the use of chemicals is based on enzymatic reactions for the formation of crosslink. The enzymes that catalyse these reactions act at neutral pH, in aqueous medium and at room temperature. This allows to exploit them to form the hydrogel in situ. In addition, unwanted or toxic side effects, which may occur with reactions mediated by photoinitiators or organic solvents, are avoided thanks to one of the best features of this type of reaction: the specificity of the enzyme substrate. The polymerization reaction can be directly controlled by modulating the enzymatic activity. Many kinds of enzymes were used:

1. Tyrosinase: it is also known as phenoloxidase and monophenol monooxy-genase and catalyzes the formation of cross-links in the absence of co-factors. It is a copper containing enzyme that catalyzes the oxidation of phenols, as in tyrosine residues and dopamine, in quinones activated, in the presence of O2. The formation of gelatin or chitosan gel takes

place in a few minutes, but they can be formed only in the presence of chitosans and are unstable and mechanically weak. More than for in vitro studies, these gels can be used as glue to accelerate healing of wounds (Wu, Bentley, and Payne, 2011).

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2. Phosphopantetheinyl transferase: the mechanism of action to form syn-thetic hydrogel consists in the transfer of a phosphopantetheine group of macromers of coenzyme A-PEG functionalized with a serine residue of the engineered carrier proteins. The hybrid hydrogels are formed by mixing the precursors of 8-arm-PEG-coenzyme A, at 37◦_{C, neutral pH}

and in the presence of M g2+_{, in about 15 minutes. This type of}

reac-tion is very attractive for studies of cell biology and tissue engineering, but it is yet to be explored (Mosiewicz, Johnsson, and Lutolf, 2010).

3. Lysyl oxidase: the lysyl oxidase is a key enzyme for the formation and repair of the native extracellular matrix. This ubiquitous enzyme oxidizes primary amines of lysines to aldehydes. These covalent bonds that are formed stabilize the fibers that form collagen and elastin. Con-sequently, this enzyme is involved in the repair of many connective tis-sues, such as skeletal, cardiovascular and respiratory tract tissue. The enzyme can be used as a cross linker for matrices and to intensify the formation of the extracellular matrix. Hydrogels become more robust thanks to continuous lysyl oxidase activity. This enzyme is abundantly present in the serum, therefore, the crosslinking of polymers contain-ing lysine occurs spontaneously without the addition of an exogenous source of the enzyme. It could be used to increase the production of extracellular matrix of the cells incorporated in the hydrogel. In addition, it can also improve the intrinsic mechanical properties of en-gineered tissue constructs and allow for the attachment of hydrogel with native tissue by the formation of covalent bonds between the rich lysine polymers of the hydrogel and the primary amines of proteins in native tissues (Bakota et al., 2010).

4. Phosphatases, thermolysin,β-lactamase and phosphatase/kinase: these enzymes may change the amphiphilicity of small peptides derived, for example, by phosphorylation mediated by kinases or

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dephosphoryla-2.5. 3D models for liver fibrosis

self-assembly and non-covalent interactions of the amphiphilic peptides in nanofibre, with consequent formation of hydrogels. The phosphatase catalyzes the removal of phosphate groups from a substrate, which be-comes hydrophobic. In an aqueous environment, these substrates can self-assemble to form a 3D nanofiber networks, through non-covalent in-teractions, which allows the formation of gel. The termolisine catalyze the formation of bonds between peptides through reverse hydrolysis. This enzyme therefore reduces the solubility of one of the peptides that can self-assemble into a hydrogel via hydrophobic interactions. The β-lactamase, produced in some bacteria, catalyzes the breakage of rings and formation of a linear molecule of 4 carbon atoms, present in the structure of lactams. When these molecules are linearized, they can self-assemble to form hydrogels (Schnepp, Gonzalez-McQuire, and Mann, 2006).

5. Peroxidases: these are enzymes that, usually, catalyze the decompo-sition of hydrogen peroxide and organic peroxides according to the following reaction: ROOR0 _{+ 2e}− _{→ ROH + R}0_{OH. Most of the used}

peroxidases have hydrogen peroxide as substrate. The peroxidases com-monly used for the formation of hydrogels are the horseradish and soy peroxidase. These enzymes are widely used, given their lack of cyto-toxicity in vivo and the speed with which they form crosslinks. (Lee, Chung, and Kurisawa, 2009)

6. Transglutaminase: These constitute a broad family of enzymes that catalyze post-translational modifications of proteins primarily by in-ducing the formation of isopeptide bonds, but also through the cova-lent conjugation of polyamines, esterification of lipids, or deamination of glutamine. They are an alternative to chemical cross-linking, as they catalyze the formation of covalent bonds between a free amino group of a protein, mainly a lysine, and the γ-carboxamide group of a glu-tamine of another protein or peptide. Once formed, these bonds are

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highly resistant to proteolytic degradation. The gels that are formed through these cross links can be used to include the cells, since they are highly biocompatible, and they have excellent transport properties, that facilitate studies of administration of drugs (Teixeira et al., 2012). Recently, the microbial transglutaminase (mTG), derived from a vari-ant Streptoverticillium mobaranse has been found to be very useful for the formation of cross- links in hydrogels. The mTG is a calcium-independent enzyme that catalyzes the formation of covalent cross-links between glutamine and lysine in proteins. In addition to the prepara-tion of hydrogels, this enzyme has many different applicaprepara-tions, also in the food industry. The cross-link formation not only depends on the presence of free residue of glutamine and lysine, but also on the tertiary structure of proteins (Broderick et al., 2005).

Figure 2.4: Transglutaminase reaction. The enzyme catalyzes the formation of covalent

bonds between a free amino group of a lysine of a protein and the γ-carboxamide group of a glutamine of another protein or peptide (Payne et al., 2013).

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Chapter 3 Aim of the Thesis

The aim of this thesis is to set up a new in vitro model to mimic liver fibrosis, by focusing on the effects of extracellular matrix stiffening on parenchymal hepatic cells. 3D culture conditions were thus optimized and the human hep-atoma HepG2 cell line was used. Cells were encapsulated in collagen gels and different parameters such as morphology, vitality and cell proliferation were evaluated. Albumin production was analysed as a metabolic functionality parameter.

Unlike most methods debated in literature, the scaffold stiffening was product with a completely biocompatible cross-linker, the enzyme microbial transg-lutaminase (mTG), which catalyzes the formation of covalent cross-links be-tween glutamine and lysine in proteins. The micromechanical properties of gels were analysed by nanoindentation and the effects of gel stiffening on cell morphology, vitality and proliferation were evaluated.

Finally, the expression of two markers of epithelial-mesenchymal transition, i.e. vimentin and E-cadherin, was assessed by western blot analysis and im-munocytochemical detection.

In the last part of this study, also the preparation of scaffolds with a de-cellularized and digested hepatic extracellular matrix, obtained from porcine liver, was taken into account.

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Chapter 4 Materials and Methods

4.1 Cell culture

The HepG2 human cell line (Interlab Cell Line Collection, IRRCS AOU San Martino, Genova (GE)), derived from a hepatocellular carcinoma was used. Cells were cultured supplemented in MEM with 10% Fetal Bovine Serum (FBS), 2mM L-Glutamine and 1% non-essential amino acids, at 37◦_{C with}

5% CO2.

4.2 3D collagen scaffolds

The 3D collagen scaffolds of HepG2 were obtained by encapsulating cells in a solution of collagen obtained from bovine skin. A 3 mg/ml stock solution was purchased by Sigma (C4243, 3 mg/ml - Sigma-Aldrich®, St. Louis, MO, USA) and gels were prepared according to the manufacturer’s instructions. Briefly, eight parts of collagen were mixed with one part of a mix composed of 50% Medium M199 (Sigma-Aldrich®, St. Louis, MO, USA) 10X and 50% FBS. The pH of the mixture was adjusted to 7.8 - 8.1 by the addition of NaOH 0.1 M and the pH was monitored with a litmus paper. The final mixture was then stored at 4◦_{C until use.}

Cells were trypsinized, centrifuged and directly resuspended into the collagen mix to a final density of 1.25 X 106 _{cells/ml. Subsequently, volumes of 40µl of}

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4.2. 3D collagen scaffolds

collagen gel-cell mix was seeded into 96-well plate and left to gel at 37◦_{C, 5%}

CO2 for one hour. This method allowed us to obtain gels with a thickness of

1 mm containing 50,000 cells. After the gelation, 150 µl of complete culture medium were added, by pulling the gels from the plate’s boards, to allow a complete and uniform diffusion of oxygen and nutrients.

Collagen + M199 medium/FBS

1.25 X 10 cells/ml

40 µl

Incubation for 1 hour at 37 °C,5% CO Gels with a thickness of 1 mm containing 50,000 cells. 150 µl of medium with mTG Lys-NH NH_-Gln

Formation of covalent bonds between a free amino group of a lysine of a protein and the -carboxamide group of a glutamine of another protein or peptide

Figure 4.1: Gelation and crosslink of a collagen gel with a thickness of 1 mm and 50,000

cells enclosed. See text for more details.

Stiffening of collagen gels

To induce the stiffening of gels, two different concentrations of microbial transglutaminase (mTG) were added to the culture media. In particular, after cells encapsulation and gelation, culture media containing 0.7 mg/ml or 7 mg/ml of mTG (and corresponding to 100 U or 1,000 U of mTG for grams of collagen, respectively) were added to gels as described above.

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4.3. Evaluation of mechanical properties

4.3 Evaluation of mechanical properties

Piuma Nanoindenter (Optics11, Amsterdam, NL) is an instrument for the characterization of the micromechanical properties of soft materials. It uses a fiber-optical sensor to gently push a spherical glass tip on the surface of the sample. For the mechanical tests, 50,000 cells were encapsulated in collagen, seeded in triplicate in a 96-wells plate and treated with mTG as described above. At 24 and 96 hours, the samples were analysed. Briefly, a probe was brought in contact with the gels surface, pushed into the material and then retracted, recording load (P) and displacement (h) over time (t). The obtained P-h datasets were analysed with the nano-epsilon-dot method as described by Mattei et al. (Mattei, Gruca, et al., 2015), obtaining the relative Young’s modulus. Piuma allows several measurements over the surface of the sample and spatial mapping of its local mechanical properties.

4.4 Trypan blue exclusion assay

Cell viability and proliferation were studied with the trypan blue exclusion test. As described above, a number of 50,000 cells were encapsulated in colla-gen gels, seeded in triplicate in a 96-wells plate and treated with mTG at the indicated concentrations. After 24, 48 and 96 hours from the encapsulation, all the gels were solubilized with the addition of a solution containing the enzyme collagenase H (COLLH-RO - Sigma-Aldrich®, St. Louis, MO, USA) dissolved in not integrated culture medium. After an incubation of 15 min-utes at 37◦_{C and 5% CO}

2, collagenase activity was inhibited by the addition

of sodium-EDTA 20 mM dissolved in Phosphate Buffered Saline (PBS). As a reference control for cell proliferation, monolayers of HepG2 (50,000 cells) were seeded in 24-wells plates and harvested by trypsinization after 24, 48 and 96 hours in culture. Finally, cells obtained from both gels and monolayers were stained with trypan blue and then were counted using a hemocytometer under a light microscopy (Leica). Trypan blue positive cells were considered dead, whereas live cells with intact cell membranes were not stained o coloured. Each assay was performed in triplicate. The numbers

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4.5. Cell viability assay: Live-Dead test

obtained were compared with the values at time zero, in order to estimate the rate of growth.

4.5 Cell viability assay: Live-Dead test

Live-Dead®Viability/Cytotoxicity Kit, for mammalian cells (L3224 - Ther-moFisher, Waltham, MA USA) is a two-colours assay used to determine cells vitality. It’s based on plasma membrane integrity and esterase activity, since it contains calcein and Propidium iodide. The non-fluorescent acetomethoxy derivate of calcein can be transported through the cellular plasma membrane into live cells where intracellular esterases remove the acetomethoxy group and gives out strong green fluorescence. As dead cells lack active esterases, only live cells are labelled. Propidium iodide, instead, is an intercalating agent and a fluorescent molecule that when it’s bound to nucleic acids re-leases red fluorescence. Propidium iodide is membrane impermeant and gen-erally excluded from viable cells, therefore, the cells that are red after the treatment are dead.

As described above, 50,000 cells for each condition were seeded in a 96-well plate (cells enclosed in collagen gels) or a 24-well plate (control monolayer). After 24, 48 and 96 hours, Live-Dead®kit solution was added to samples me-dia according to manufacturer’s instruction and incubated for 20 minutes at room temperature. Gels were then transferred onto glass microscope slides, covered with a coverslip and analysed under a fluorescence microscope (Leica DMR). Also, cell monolayers - used as positive control - where trypsinized and transferred onto glass microscope slides. Two suitable filters were then used to visualize calcein and Propidium iodide positive cells.

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4.6. Cell functionality assay: Albumin secretion

4.6 Cell functionality assay: Albumin

secre-tion

The concentrations of albumin secreted from cells encapsulated in the scaf-folds and from monolayers were determined using a human albumin enzyme-linked immunosorbent assay (ELISA) Quantitation kit (RAB0603- Sigma-Aldrich®, St. Louis, MO, USA) according to the manufacturer’s instruc-tions.

As described above, 50,000 cells for each condition were seeded in a 96-well plate (cells enclosed in collagen gels) or a 24-well plate (cells on monolayer). After 24, 48 and 96 hours, the culture media from both gels and monolayer, were centrifuged at 1.71 g for 5 minutes and the supernatants were stored at −20◦_{C. Briefly, the assay was performed in 96-well plates coated with a}

cap-ture antibody. A standard curve was prepared by using an albumin solution supplied by the manufacturer; 1:2.5 serial dilutions were then made in order to prepare standard solutions ranging from 12,000 to 49.17 pg/ml of albumin. Assay Diluent Buffer was used as zero standard (0 pg/ml). Culture media samples and standard solutions were then added to the plate (100 µl). The plate was incubated overnight at 4◦_{C with gentle shaking. The next day, the}

solution was discarded and the plate was washed four times with 1X Wash Solution. At every wash, the plate was inverted and blotted against a clean paper towel. After the last wash, the remaining Wash Buffer was removed by aspirating. Subsequently, 100 µl of Biotinylated Detection Antibody, diluted 80-fold with 1X Diluent Buffer, were added to each well and the plate was incubated for one hour at room temperature with gentle shaking. The solu-tion was then discarded and the plate was washed four times. A volume of 100 µl of HRP- Streptavidin, diluted 2,000-fold with 1X Diluent Buffer, was then added to each well, and the plate was incubated for 45 minutes at room temperature with gentle shaking. The incubating solution was then removed and, after other four additional washes, 100 µl of ELISA Coulometric TMB Reagent were added to wells, and the plate was incubated for 30 minutes at room temperature, in the dark, with gentle shaking. Finally, 50 µl of

A 3D in-vitro model of hepatic fibrosis for biomedical applications

Universit`

a degli Studi di Pisa

Tesi di laurea magistrale

A 3D in-vitro model of hepatic fibrosis for

biomedical applications

Contents

Abbreviations

Chapter 1

Abstract

Chapter 2

Introduction

2.1

The Liver

2.1.1

Liver cells

2.1.2

Extracellular matrix

2.1.3

Wound repair and scar forming

2.2

Liver Fibrosis

2.2.1

Hepatic myofibroblasts

2.2.2

Hepatic stellate cells

2.2.3

Portal fibroblasts

2.2.4

Bone-marrow-derived fibrotic cells

2.2.5

Fibrogenic cells from Epithelial-Mesenchymal

Tran-sition

2.3

Animal models for liver fibrosis

2.3.1

Immunologically mediated fibrosis

2.3.2

Cholestatic fibrosis

2.3.3

Chemically induced fibrosis

2.3.4

Transgenic animals

2.4

In vitro

models for liver fibrosis

2.4.1

Hepatic stellate cells

2.4.2

Precision-cut liver slices

2.4.3

Parenchymal cells

2.5

3D models for liver fibrosis

2.5.1

Hydrogels

2.5.2

Scaffolds

2.5.3

Spheroids

Chapter 3

Aim of the Thesis

Chapter 4

Materials and Methods

4.1

Cell culture

4.2

3D collagen scaffolds

4.3

Evaluation of mechanical properties

4.4

Trypan blue exclusion assay

4.5

Cell viability assay: Live-Dead test

4.6

Cell functionality assay: Albumin

secre-tion