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In Vitro Intestinal and Liver Models for Toxicity Testing Sophia M Orbach, Rebekah R Less, Anjaney Kothari, and Padmavathy Rajagopalan ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00699 • Publication Date (Web): 03 Jan 2017 Downloaded from http://pubs.acs.org on January 7, 2017

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ACS Biomaterials Science & Engineering

In Vitro Intestinal and Liver Models for Toxicity Testing

Sophia M. Orbach1, Rebekah R. Less2, Anjaney Kothari2, Padmavathy Rajagopalan1,2,3,*

1

Department of Chemical Engineering, Virginia Tech, Blacksburg, Virginia 24061, United States 2

School of Biomedical Engineering and Sciences, Virginia Tech, Blacksburg, Virginia 24061, United States

3

ICTAS Center for Systems Biology of Engineered Tissue, Virginia Tech, Blacksburg, Virginia 24061, United States

* Corresponding author email: [email protected] Mailing address 333 Kelly Hall 325 Stanger St Blacksburg, VA 24061

1

ACS Paragon Plus Environment


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Abstract The human body is exposed to hundreds of chemicals every day. Many of these toxicants have unknown effects on the body that can be deleterious. Furthermore, chemicals can have a synergistic effect, resulting in toxic responses of cocktails at relatively low individual exposure levels. The gastrointestinal (GI) tract and the liver are the first organs to be exposed to ingested pharmaceuticals and environmental chemicals. As a result, these organs often experience extensive damage from xenobiotics and their metabolites. In vitro models offer a promising method for testing toxic effects. Many advanced in vitro models have been developed for GI and liver toxicity. These models strive to recapitulate the in vivo organ architecture to more accurately model chemical toxicity. In this review, we discuss many of these advances, in addition to recent efforts to integrate the GI and the liver in vitro for a more holistic toxicity model.

Keywords First-Pass Metabolism, Gastrointestinal, Hepatic, Biotransformation

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Introduction The human body is exposed to a wide range of chemicals, toxins, drugs and pharmaceuticals1. Some of the compounds intended for medicinal purposes can elicit beneficial outcomes at therapeutic doses, however higher concentrations can have detrimental effects. Exposure to drugs and chemicals can occur through inhalation, ingestion or absorption through the skin1, with oral exposure being the most prevalent form2. Overexposure to chemicals or interactions between drugs can have adverse effects on the body’s immune, nervous, digestive, respiratory, and other systems at high concentrations3-6. Therefore, there is a need to understand how such entities may affect function at the organ- and organism-level7. Traditionally, testing the toxic effects of chemicals has relied on large-scale animal studies8. In addition to being extremely expensive, there are also ethical concerns with animal studies. Due to the associated issues with animal studies, there is a need to design in vitro organotypic cultures to investigate toxicity9.

Orally

administered

drugs

and

chemicals

are

primarily

metabolized

by

the

gastrointestinal (GI) tract and the liver (Figure 1A)10. The gut is one of the primary organs exposed to orally administered drugs or chemicals11. When a xenobiotic interacts with the small intestine, it can damage the organ while also being metabolized through the actions of biotransformation enzymes12-13. Reactive metabolites formed as a result of metabolism can further damage intestinal tissue11, 13. A fraction of the chemical and its metabolites get transported across the gut epithelium and absorbed into the blood stream before reaching the liver by the portal vein14.

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Figure 1: GI tract and liver. A) A schematic representation of the GI tract and the liver depicting their orientation in the body B) macroscopic view of the path taken by chemicals and nutrients through enterohepatic circulation C) arrangement of intestinal mucosal cells in crypts and villi D) arrangement of hepatic cells along the liver sinusoid.

In the liver, biotransformation enzymes such as the class of cytochrome P450 (CYP) enzymes further metabolize chemicals and xenobiotics15. The metabolism of a chemical or xenobiotic can result in hepatotoxicity16. Chemicals and xenobiotics can undergo phase I and phase II metabolism in the liver before being safely excreted by the kidneys or secreted back into the intestines through the bile duct14. Phase I reactions include oxidation, reduction and hydrolysis7,

17-18

. The majority of phase I reactions occurs

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through the actions of the cytochrome P450 (CYP) class of enzymes. Phase II reactions include the conjugations of drugs and chemicals to glucose, sulfate, glutathione, amino acids, and acetyl or methyl groups17-18. Once the intestine and the liver have metabolized a drug or chemical, the un-metabolized fraction as well as some of the byproducts enter systemic circulation or are directed to the kidneys for excretion. Additionally, they may re-enter the intestine via the bile duct14,

19

. This process is known as enterohepatic

circulation (Figure 1B)20-21. Drugs and toxicants can remain in the enterohepatic circulation for extended periods of time due to this recirculation process resulting in increased toxicity and decreased bioavailability22.

The combined actions of the small intestine and liver affect the absorption and bioavailability of drugs, bile acid homeostasis and urea cycle14, 23-24. As a result, other organs may be affected. Because of the complex interactions between the GI tract and liver and the subsequent effects these interactions have on other organs, we focus on engineered models of the intestine and the liver and their applications in evaluating toxicity. In subsequent sections of this review, the small intestine and GI tract are used interchangeably.

Cellular Composition and Functions of the GI tract and Liver The GI tract is primarily responsible for the digestion and absorption of food and other nutrients25. The upper GI tract includes the mouth, esophagus and stomach and is responsible for the intake, transit and digestion of food. The lower GI tract is comprised of the small and large intestine (colon). The small intestine is responsible for absorption, transformation and digestion. The colon is primarily responsible for reabsorption of water

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and electrolytes from the luminal contents. The small intestine is divided into three sections: duodenum, jejunum and ileum26. Both the small and large intestines exhibit different properties across the length of the organ6, 22, 27-28.

Radially, the GI tract has 4 distinct tissue layers: mucosa, submucosa, muscularis externa and serosa/adventitia26. The mucosa is the innermost layer, exposed to the intestinal luminal contents and is the most important layer of the intestine for absorptive and secretory functions26. The mucosal epithelium is constituted of four major cell types: enterocytes, goblet cells, Paneth cells and enteroendocrine cells. These cells are arranged in invaginations known as the crypts of Lieberkühn and projections known as villi (Figure 1C)25.

Enterocytes are absorptive cells that constitute about 90% of mucosal cells29. Enterocytes express both phase I and phase II enzymes19. For example, phase I enzymes such as CYP1A1, 2C9, 2C19, 2E1, and 3A4 are expressed in human enterocytes30-31. The other major epithelial cell types are secretory in nature. Goblet cells secrete mucin proteins, which form a protective barrier. Goblet cells constitute 8-10% of the mucosal cell population29. Enteroendocrine cells secrete digestive hormones such as secretin, incretin, glucagon-like peptides 1 and 2, and serotonin and constitute less than 1% of gut epithelial cells29, 32. Paneth cells secrete antimicrobial proteins and peptides such as lysozyme and cryptdins (mammalian defensins)33. In addition, the crypts house stem cells that periodically renew the intestinal epithelial layer34.

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The liver is the largest solid organ in the body and responsible for many physiological functions35.

These

include

the

metabolism

of

glucose,

lipids,

vitamins,

and

carbohydrates, protein storage and synthesis, bile synthesis, and biotransformation of xenobiotics18,

35

. Metabolism of chemicals occurs through phase I and phase II

biotransformation enzymes7, 18. Phase I reactions commonly result in formation of toxic intermediates7, 17-18. Phase II reactions result in the excretion of the toxic substance from the body. Liver toxicity is determined by the rate of formation of toxic metabolites from phase I reactions and the corresponding rate of elimination by phase II reactions7.

Hepatocytes are the parenchymal cells of the liver and responsible for the majority of liver functions7,

35-39

. These cells are oriented into cords separated by blood vessels

known as sinusoids (Figure 1D). Bile is secreted by hepatocytes through the apical membrane into bile canaliculi located between hepatocyte tight junctions35, 38-39. Microvilli on the hepatocytes extend from the basal membranes into a protein-rich interface known as the Space of Disse35,

40-41

. This region is composed of extracellular matrix (ECM)

components, primarily fibronectin and types I and III collagen38. The Space of Disse acts as a physical barrier between hepatocytes and liver non-parenchymal cells (NPCs) and promotes the transfer of nutrients and oxygen between the sinusoid and the hepatocytes17, 35, 38.

There are three primary NPCs in the liver. They are liver sinusoidal endothelial cells (LSECs), Kupffer cells (KCs) and hepatic stellate cells (HSCs)17, 35-36, 38. LSECs line the sinusoid and prevent direct contact between blood and the hepatocytes. LSECs differ from other endothelial cells by their lack of basement membrane, presence of fenestrae

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(100-150 nm pores) and scavenging abilities17, 35-36, 38-39. The KCs are liver macrophages and are usually found in the sinusoid7, 17, 35-36, 38-39. HSCs are located in the Space of Disse and store retinoid and lipids in a healthy liver35, 38.

Upon exposure to drugs and chemicals, NPCs become activated18, 38. LSECs contract, become defenestrated, form a basement membrane, and lose their scavenging abilities18, 38, 42. KCs secrete pro- and anti-inflammatory cytokines and recruit neutrophils to the liver18,

38, 43

components18,

. Activated HSCs become myofibroblastic and secrete excess ECM

35, 38, 44

hepatotoxicity18,

. NPCs exert both protective and inflammatory roles during

38

. These cells are exposed to chemicals and xenobiotics prior to

hepatocytes. Such exposure results in the secretion of anti- and pro-inflammatory cytokines as well as reactive oxygen species (ROS)18, 36, 38.

Tissue-Engineered Models for Toxicity Evaluation GI Models In this review, we describe GI models that emulate the small intestine used primarily in toxicity evaluations. Intestinal models have been developed to investigate drug discovery, development and metabolism while addressing the accompanying toxicity45-47.

Immortalized Cell Lines In vitro GI models commonly use immortalized or cancerous intestine-like cells or cell lines48-50. Cell lines used for modeling intestinal transport and toxicity include Caco-2,

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HT-29, T84, TC-7, and SW62051-54. Among these cell types, Caco-2 and HT-29 cells are used extensively as substitutes for enterocytes and goblet cells, respectively54-55.

Caco-2 cells are derived from human colon carcinoma and have been shown to form a polarized enterocyte-like monolayer after 21 days in culture on Transwell® inserts48, 56. Polarized Caco-2 monolayers are characterized by the presence of villi, brush border enzymes and drug transporters55. Caco-2 cells can be cultured on a variety of biomaterials including alginate, collagen, polycarbonate, polystyrene, and Biocoat®55-59. This versatility makes them extremely appealing for use in engineered in vitro models. However, such cultures also have disadvantages. In terms of xenobiotic transport modeling, Caco-2 monolayers have an approximately 100-fold decreased permeability compared to the human small intestine49, 60. Furthermore, Caco-2 monolayers have an approximately 20-fold increase in organic anion-transporting polypeptide and breast cancer resistance protein transporter expression as compared to the human small intestine61-62.

Additionally,

the

expression

of

critical

phase

I

and

phase

II

biotransformation enzymes is significantly lower in such cells as compared to in vivo48-49, 60

. HT-29 cells are also derived from human colon carcinoma and can express mature

mucin proteins63. Co-culture of these goblet-like cells with Caco-2 cells results in the presence of a mucus layer and overall increased permeability, thus more accurately mimicking the in vivo microenvironment50, 54.

Caco-2 monocultures or co-cultures with HT-29 cells are frequently cultured on the apical side of a Transwell® insert to mimic the intestinal barrier54-55. Transwell® systems have been commonly used to assess the effect of environmental chemicals and

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pharmaceuticals49. The permeability of a drug or chemical is measured by determining the rate of transport across the monolayer53. The viability of the cultures has been assessed by tight junction degradation through trans-epithelial electrical resistance (TEER) measurements, changes in mitochondrial metabolism and cell viability, production of ROS, and changes in protein synthesis48, 64-65.

Intestinal Organoids Intestinal organoids have been gaining attention as a means of successfully mimicking the intestinal architecture and cellular composition (Figure 2A)66. Organoids have been assembled by embedding isolated intestinal crypt and villus units or stem cells in Matrigel™67-68. Organoids assembled from the mouse small intestine consist of all the major cell types of the GI epithelum68. Human colon derived intestinal organoids are mostly assembled with stem cells that need to be differentiated further to obtain mature intestinal cells69. Intestinal organoids can potentially find applications in patient-specific drug design, treatments and in the investigations into GI-related diseases66. However, measuring the transport and diffusion of drugs across a monolayer of intestinal epithelial cells cannot be conducted with these in vitro models70-71. Recently, Schweinlin et al. engineered small intestinal organoids using a decellularized small intestinal submucosa as a scaffold70. CYP3A4 and P-glycoprotein (a drug transporter) levels were measured and compared between organoids cultured alone or on the scaffolds70. Both CYP3A4 and P-glycoprotein levels were more than three-fold higher in organoids cultured on scaffolds in a bioreactor than organoids in a static system. However, intestinal organoids have not been thoroughly explored for drug toxicity assessment.

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Explants and Everted gut sacs Everted intestinal sacs are ex vivo systems used to primarily study drug absorption and transport72. By everting the intestinal tubes, the mucosal surface is directly exposed to the environment72-73. Everted intestinal sacs from the small intestine have been used to study the transfer, absorption and metabolism of drugs such as acetaminophen (pain reliever), midazolam (sedative), testosterone (hormone), bupropion (antidepressant), and dextromethorphan (cough suppressant)74-75. Absorption and metabolism of herbal medicinal compounds, such as paeoniflorin and sinomenine, have also been investigated using everted sacs76. Paeoniflorin was not metabolized by the everted gut sacs similar to observations in vivo, and its absorption increased 2.5-fold in the presence of sinomenine76-77.

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Figure 2: Models of GI toxicity. A) An intestinal organoid including the primary cells in the mucosal epithelium B) PCIS showing the maintenance of the layers in the intestinal wall and C) gut-on-a-chip model.

While everted sacs are usually maintained only for a few hours, traditional explant cultures enable longer investigation78-81. Explants from the small and large intestines have been cultured from a few days up to weeks82. Explants range from full thickness explants (mucosa to serosa)82 to only mucosa83-84 to biopsies78-79, 85-86.

To promote explant adherence and maintenance of phenotype, different substrates have been explored87. These include glass fiber paper, steel grids, sponges, Transwell® inserts, and cellulose acetate filters78, 81, 83-84, 86, 88-90. Such models have been used to

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study toxicity and CYP expression. For example, jejunal explants have been used to study regulation of CYP3A80 . Dexamethasone-induced CYP3A1 in jejunal biopsies was qualitatively similar to in vivo as seen through Northern and Western blots. In another study, effects of deoxynivalenol, a contaminant in cereals and grains, were investigated using jejunal explants81. Environmentally relevant concentrations of this toxin caused toxicity in the explants, that had previously been shown only in mice in vivo81.

Precision cut intestinal slices (PCIS) represent a class of intestinal explants that differ from the traditional explants in their thickness. These are 300-400 µm thick tissue slices obtained from agarose-filled and -embedded intestinal tissues cut using precise tissue slicers (Figure 2B)91. PCIS have been investigated in drug metabolism and toxicity studies92. Specifically, these cultures have been used to study diclofenac (non-steroidal anti-inflammatory drug) metabolism and toxicity93-94. Diclofenac, and not its metabolites, was found to lead to intestinal toxicity in PCIS93. In another study, Martignoni et al. found comparable induction of most CYP enzymes between intestinal sections in vivo and PCIS in vitro95.

A Comparison of Current Intestinal Models Cultures containing immortalized intestinal cell lines can be used to investigate the transport of drugs as well as in toxicity testing. However, the concentration of drugmetabolizing enzymes is very different from those measured in the GI tract48-49,

70

.

Intestinal organoids exhibit significant potential in studying development and disease66. However, such cultures can take a long time before mature intestinal cells are obtained. In addition, traditional measurements on drug transport and permeability cannot be

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accurately recapitulated in such models70-71. While everted sacs can only be used for short term studies lasting a few hours, explants can be cultured for longer time periods lasting a few days72-73, 78, 80. However, lack of consistency across explant culture poses a major challenge for toxicity studies96.

Liver Models In vitro liver toxicity models frequently utilize mouse or rat primary hepatocytes36,

38

.

These species are preferred due to their low sentience, cost, easy accessibility, and the ability to compare in vitro results to data obtained from the corresponding animal97-99. Difficulties arise when the toxic response of animal models are extrapolated to human hepatotoxicity98. Mice are typically several-fold more sensitive to toxicants than humans, whereas rats are known to be less sensitive38,

100

. Therefore, choosing appropriate

species and relevant comparisons are critical to interpreting accurate information from in vitro hepatotoxicity studies98.

While most hepatotoxicity evaluations are conducted with primary cells, immortalized human hepatocyte-like cell lines including Fa2N-4, HepG2, C3A, Huh7, and HepaRG cells are also utilized36, 38. HepG2 cells are the most commonly used human liver cell line and are derived from human hepatocarcinoma36. However, the expression of phase I and phase II biotransformation enzymes is greatly reduced in HepG2 cells relative to primary human hepatocytes36, 101. Specifically, expression of CYP enzymes in HepG2 cells is decreased up to 50-fold101 HepaRG cells are also derived from hepatocellular carcinomas, but differentiate into two distinct cell populations when cultured in monolayer97, 102. One cell type, that can be preferentially induced by dimethylsulfoxide, is

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similar to hepatocytes, while the other resembles cholangiocytes36. The expression of CYP enzymes in these cells varies within 10-fold of primary hepatocytes, making HepaRG cells the preferred cell line for toxicity testing36, 101, 103.

More recently, induced pluripotent stem cells (iPSCs) have been utilized as a self renewing source of human hepatocytes36. Somatic cells can be obtained from specific individuals, which could enable patient-specific studies of idiosyncratic hepatotoxicity38, 104

. iPSCs can be differentiated into hepatocytes by the addition of medium supplements

such as activin A, fibroblast growth factor ,Wnt3a, insulin, dexamethasone, hepatocyte growth factor, and oncostatin M36, 2D36,

106-108

and 3D36,

109-111

97, 105

. Hepatocyte-like iPSCs have been utilized in

liver models for toxicity testing. There are two major

disadvantages with the use of hepatocyte-like iPSCs. The first is that the expression of biotransformation enzymes resembles that of fetal hepatocytes, rather than mature hepatocytes36. However, a recent study has demonstrated that a 3D configuration and the addition of cyclic adenosine monophosphate can address these issues112. The second major disadvantage is that there is substantial variability between cell lines as a result of the source and differentiation protocols97.

Since primary hepatic cells are highly metabolic and can rapidly dedifferentiate in culture, accurate measures of hepatotoxicity are often difficult36,

38, 113

. In subsequent

sections, we provide background on traditional 2D hepatic cultures and more advanced 3D organotypic models.

Hepatotoxicity Testing in 2D Models

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Hepatocyte monolayers are assembled by seeding hepatocytes on protein-based substrates (Figure 3A)113-114. In such cultures, hepatocytes rapidly lose hepatocytespecific functions and expression of biotransformation enzymes within 24-72 h of seeding113,

115

. For these reasons, hepatocyte monolayers are less sensitive to

chemicals compared to the native organ resulting in a significant underestimation of toxicity116-119. Despite such inaccuracies, hepatocyte monolayers are still commonly used for toxicity studies100, 116-119.

Figure 3: 2D models of liver toxicity. A) Hepatocyte monolayer B) collagen sandwich C) co-culture of hepatocytes with other cell types including NPCs, endothelial cells, fibroblasts, and other epithelial cells

In collagen sandwich models (Figure 3B) the continued expression of CYP enzymes leads to greater sensitivity to xenobiotics100, 120-126. Despite the benefits of the sandwich model, it does not incorporate heterotypic intercellular interactions that occur in the liver. The co-culture of hepatocytes with other cell types enables heterotypic cell-cell interactions (Figure 3C)127. These cells include NPCs, endothelial cells, fibroblasts, and other epithelial cells. Hepatocyte phenotypes can be maintained up to five weeks in culture when additional cell types are added within a week of seeding127-129.

Expression of CYP enzymes are up to two-fold higher in co-cultures relative to hepatocyte monolayers130. These cultures have been utilized for toxicity studies and

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exhibit increased function but comparable sensitivity to hepatocyte monolayers107-108, 131134

. This is particularly true for acetaminophen treatment, since CYP2E1 expression is

not maintained in 2D co-cultures130-131, 133. However, Nelson et al. found that C3A/human vascular endothelial cell co-cultures exhibited increased ATP in response to acetaminophen relative to monocultures of either cell type107. This indicates that hepatocyte-like cell lines do not respond to 2D co-culture similarly to primary cells. Micropatterning of co-cultures has been developed to better replicate in vivo cellular organization in a 2D configuration36,

38

. These models control the heterotypic inter-

cellular interactions, support the differentiation of iPSCs to hepatocytes and have demonstrated improved sensitivity relative to standard 2D co-cultures108, 135-139.

Hepatotoxicity Testing in 3D Models 3D liver models have been shown to maintain hepatocyte phenotype and biotransformation enzyme expression36, 38. These models emulate in vivo properties of the liver such as heterotypic intercellular interactions, the stratified architecture, maintenance of hepatocyte tight junctions, induction of polarity, kinetic flow, and the presence of ECM components7, 17-18, 35-39, 127, 140. The recapitulation of these properties prevents dedifferentiation and maintains CYP expression and chemical sensitivity over extended periods of time36,

38, 140

. For these reasons, 3D liver models have become

regularly utilized for the investigation of in vitro hepatotoxicity36, 38.

Precision cut liver slices (PCLS) are 100-250 µm thick sections of whole liver that can be cultured in vitro for a maximum of 72 h due to restrictions in oxygen and nutrient diffusion (Figure 4A)38. However, gene expression of PCLS varies drastically after 24 h

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in culture141. Diffusion is a critical limiting factor in toxicity studies, since chemicals cannot reach all the cells38. Administration of PCLS with acetaminophen results in protein profiles similar to in vivo and predicts relevant toxic concentrations141-143. Unlike many other in vitro models, the microarchitecture, intercellular interactions and CYP expression are maintained from in vivo142, 144-145. Specifically, CYP expression accurately depicts inter-donor variations from human livers145.

Figure 4: 3D models of liver toxicity. A) PCLS depicting the maintenance of hexagonal liver lobules and the location of the portal triad and central vein B) hepatocyte spheroids in a culture well C) hepatocytes in a bioreactor system.

PCLS models can be used to analyze the role of individual cell types on overall liver toxicity. For example, KCs can be depleted in vivo by gadolinium chloride146-147. PCLS from gadolinium chloride treated rats predict decreased tumor necrosis factor alpha

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secretion and acetaminophen toxicity upon KC depletion146. Analyzing protein profiles in PCLS are useful for detecting metabolites and protein adducts144,

146

. However, the

proteins upregulated in response to a chemical are not always accurate, as the corresponding predicted mechanisms of cell death do not always match in vivo143.

Spheroids are small cell aggregates formed through numerous techniques such as the hanging-drop method, seeding on non-adhesive surfaces and rotating cultures (Figure 4B)36,

38

. Spheroids are the most commonly used 3D liver models for hepatotoxicity

studies and, unlike PCLS, can maintain biotransformation enzymes for up to five weeks109, 148-154. Unlike other liver models, hepatocytes in spheroids become increasingly more sensitive to chemicals after several days in culture due to increased interactions between the cells over time148, 151, 153, 155. Sensitivity of the spheroids to chemicals can also be dependent on the surface used to assemble the model150. For instance, spheroids formed on MatrigelTM are more resilient to nanoparticle toxicity than those seeded on collagen. Cells on the outside of spheroids are more sensitive to chemicals than those in the center110, 156. Therefore, increases in the diameter of spheroids can be used as a marker of toxicity110.

Hepatocyte spheroids respond to acetaminophen similarly to in vivo as seen through cell death, mitochondrial damage, glutathione depletion, and response to N-acetylcysteine149, 151, 157

. CYP enzymes are consistently better induced in spheroids than 2D models109, 153-

154

. Particularly, CYP2E1 is maintained in spheroids for up to three weeks, the isoform

not expressed in 2D co-cultures130,

148, 151

. Therefore, when treated with some

compounds, spheroids can be less sensitive than 2D models110,

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151, 154, 158

. However,


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these responses vary from study to study depending on the type of cell used and method of spheroid formation.

Hepatocytes can be encapsulated in hydrogels to induce polarity and extend hepatocyte function159-163. The addition of acetaminophen to hepatocytes in gel entrapped hollow fibers can increase toxicity up to four-fold relative to monolayers162-163. Encapsulation of hepatic cells in alginate hydrogels does not always improve chemical sensitivity relative to 2D models159-160. For example, encapsulated HepG2 cells are more responsive to acetaminophen and diclofenac than monolayers, but are comparably sensitive to quinidine (antiarrhythmic drug) and rifampicin (anti-tuberculosis drug)160.

Investigating Hepatotoxicity Under Dynamic Flow Bioreactors and liver-on-chip devices incorporate kinetic flow to emulate vascular flow. In vivo, vascular flow replenishes nutrients, provides oxygen and removes cell debris and toxic byproducts (Figure 4C)36, 38. When culture medium is circulated in a device or a bioreactor, it also enables the ability to investigate the response of multiple chemicals and concentrations36. Hepatocytes in devices with flow exhibit increased CYP expression and functional markers, such as albumin and urea, relative to static cultures138, 155, 164-166.

The incorporation of spheroids and scaffolds within bioreactors improves hepatocyte functions and CYP expression making them suitable for toxicity evaluations152, 155, 164, 167173

. Their inclusion in bioreactors results in biotransformation more comparable to in vivo

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and can maintain liver function up to seven weeks152,

167, 169, 171, 174-178

. Hepatocyte

spheroids in a bioreactor can maintain significant toxicity to chemicals up to three weeks in culture169,

177

. Such chemicals include acetaminophen, amiodarone (antiarrhythmic

drug), diclofenac, metformin (diabetes medication), phenformin (diabetes medication), and valproic acid (anti-epileptic drug)169. Additionally, after three weeks in culture, the number of CYP enzymes that maintained in vivo-like expression in kinetic models was four-fold higher than static models177.

In many bioreactors, hepatocyte toxicity (relative to static cultures) is reduced as a result of the removal of toxic byproducts168-169,

171-173

. Depending on the relevance of these

metabolites to the mechanism of toxicity, this can lead to results that either converge or diverge from in vivo. For example, hepatocytes in a bioreactor have exhibited a LC50 of 40 mM for acetaminophen in rats, approximately twice that of in vivo169, 174. However, the inclusion of J2-3T3 fibroblasts in this culture has been shown to increase the sensitivity to acetaminophen up to three-fold174. This demonstrates that in a bioreactor, the inclusion of heterotypic inter-cellular interactions results in a system more representative of in vivo and can overcome the loss of critical metabolites as a result of the flow.

Hepatocytes have been cultured in microfluidic devices to assemble liver-on-a-chip systems36,

38, 138, 155, 179-181

. Hepatic cells can be cultured in a 2D conformation or

encapsulated in ECM proteins as a ‘micro-tissue’137.Toxicity can be measured in these systems via fluorescence visualization138, 155, 164-166, 175, 179, 181. This analysis provides both temporal and spatial variations in toxicity within the culture. Fluorescence visualization can be used to quantify live, dead and apoptotic cells138, 155, 164, 179, 181, identify cell types,

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organization and organelles164, 166, 175, detect changes in protein expression155, 165-166, 175, and establish hepatic cell activation166 in response to chemicals.

Liver-on-a-chip studies have also been conducted using immortalized hepatocyte-like cell lines155, 164-165, 179, 181. Spheroidal cultures of HepG2 cells and NIH-3T3 fibroblasts in a microfluidic device have demonstrated some apoptosis at low concentrations of acetaminophen and extensive necrosis at 20 mM179. Necrosis as the primary mechanism of acetaminophen hepatotoxicity is consistent with in vivo studies182. Similarly, HepG2/C3A spheroids in microfluidic devices exhibited significant acetaminophen toxicity at 15 mM155. When HepG2 cells were cultured on a scaffold in a microfluidic device, they exhibited an approximately 50% decrease in viability at 50 mM ethanol (alcohol), approximately three-fold lower than the LC50 reported in humans181, 183. These results demonstrate that despite the reduced biotransformation capacity in HepG2 cells, their inclusion in microfluidic devices greatly increases the sensitivity of the cells.

There are several liver-on-a-chip models designed to recapitulate the hepatocyte cords and sinusoidal architecture184-187. In these models, cylindrical cell chambers are separated from medium perfusion chambers by LSEC-like barriers to emulate fenestrae. Such a design provides a constant influx of nutrients and oxygen while preventing shear stress to the hepatocytes185-186. A variety of chemicals have been tested with these types of models including diclofenac, acetaminophen, isoniazid (anti-tuberculosis drug), rifampicin, quinidine, and ketoconazole (anti-fungal)184-187. The majority of these treatments resulted in increased sensitivity in 3D models relative to 2D conformations185186

. The predicted LC50 of acetaminophen, rifampicin and ketoconazole in the 3D models

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were similar to those reported in vivo186. However, the estimated LC50 for diclofenac and quinidine were approximately three-fold lower than in vivo. The increased toxicity of such chemicals in vitro is most likely due to the critical role of enterohepatic circulation in the metabolism of the drugs in vivo18.

Comparing the Current State of the Art of Hepatotoxicity Models The hepatocyte monolayer is the simplest liver model to assemble, but cells quickly dedifferentiate and lose their ability to metabolize chemicals. Collagen sandwich models and 2D co-cultures exhibit sensitivity to toxins for several weeks. However, the collagen sandwich culture lacks the necessary heterotypic intercellular interactions and 2D cocultures do not accurately mimic the stratified hepatic microenvironment. PCLS include all liver cell types but exhibit only a limited viability. Spheroids and encapsulated hepatocytes improve intercellular and cell-matrix interactions, respectively. However, both models are diffusion limited and do not emulate the in vivo cellular structure. Therefore, different hepatic models may be used based upon the types of toxicity evaluations to be conducted.

Integrated Intestinal-Liver Models for Toxicity Evaluation Modeling the enterohepatic circulation and first pass metabolism in vitro is essential for accurate predictions of drug bioavailability and toxicity in vivo188. In an attempt to recapitulate the integration of the GI tract and the liver, several in vitro models have been established.

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Static Transwell® Integrated Models Choi et al. first developed a static integrated Caco-2/HepG2 system to study toxicity189. This model utilizes a Transwell® insert with Caco-2 cells cultured on the apical side and HepG2 cells cultured on the basal side of the membrane. This architecture is designed to mimic the sequence of oral absorption and metabolism of xenobiotics in vivo. Integration of Caco-2 cells with HepG2 cells results in increased CYP1A2 activity and decreased acid phosphatase activity after treatment with benzo[a]pyrene, a carcinogen found in food products. These results suggest increased sensitivity when an intestinelike model is integrated with a liver-like model. Similar models have been used to investigate first pass metabolism, bioavailability and toxicity of pharmaceuticals undergoing clinical trials188.

Microfluidics-based models In an attempt to model the dynamic nature of first pass metabolism, several models have been developed incorporating intestinal and hepatic models in fluidic systems to monitor drug transport, metabolism and toxicity (Figure 5A)81, 189-195. Mahler et al. developed an integrated microfluidic model incorporating an intestinal chamber (co-culture of Caco2/HT29-MTX cells) with a liver chamber (HepG2/C3A cells)194. This model incorporated flow rates scaled from in vivo for both the intestine and liver. After treatment with acetaminophen, the microfluidic model resulted in a 40% increase in drug metabolism and 50% decrease in intracellular glutathione content as compared to static control. Similar microfluidic systems have been used to predict the absorption, metabolism, toxicity, and efficacy of anti-cancer drugs191. Incorporation of intestine-like and liver-like chambers resulted in a 30% decrease in cell viability after treatment with the anti-cancer

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drugs 5-fluorouracil and Tegafur compared to organ-specific models. This was due to increased CYP activity after integration of the intestine- and liver-like models.

Figure 5: Integrated GI-liver models of toxicity. A) An integrated microfluidics model incorporating intestinal and liver cells maintaining the in vivo exposure order of the organs to chemicals B) an integrated gut-liver model using PCIS and PCLS.

PCIS and PCLS Integrated Model Very few studies have integrated primary cells or tissues from the gut and the liver for drug metabolism and toxicity studies196. Recently, PCLS and PCIS have been used to develop integrated gut-liver systems from primary tissues (Figure 5B)197. This system uses a microfluidic chip to direct fluid flow from the PCIS chamber to the PCLS chamber. While this system was not used to investigate drug metabolism or toxicity, interplay between the two organs was demonstrated by regulation of bile acid synthesis.

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Page 26 of 51

Integration of these slices also resulted in the down-regulation of CYP7A1, responsible for bile acid regulation198. Furthermore, the induction of phase I and phase II enzymes were validated by 7-ethoxycoumarin, 7-hydroxycoumarin, and lidocaine (nerve block). These results suggest that fluidic precision cut slice models could be utilized for modeling first pass metabolism and drug toxicity in vitro.

Future Directions The development of accurate in vitro GI and liver toxicity predictive models is still actively being pursued. The overall goal of these models is to more accurately mimic the in vivo microenvironment and corresponding toxicity predictions. Several models developed

to

recapitulate

this

microenvironment

have

shown

preliminary

biotransformation capabilities, but have not yet been characterized for toxicity testing.

Future Intestinal Toxicity Models 3D intestinal models have recently been recently been developed in an effort to model the villi structure of the small intestine59. These models utilize a collagen or alginatecoated hydrogel (polymethylmethacrylate or polydimethylsiloxane) as 3D substrates for Caco-2 cells. This results in a polarized monolayer after 14 days in culture, as compared to 21 days in standard Transwell® culture. Furthermore, 3D models exhibit decreased TEER values, increased mucin production and increased drug permeability compared to static Transwell® models2. In an attempt to more accurately model the dynamic nature of the GI tract, intestinal cell lines have been cultured in microfluidic devices (Figure 2C)199. The incorporation of flow and peristaltic-like motions has been shown to decrease the permeability of intestinal cell monolayers, maintain brush border enzymes

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and increase mucin production compared to static cultures200. While these models are only recently developed and have not been used extensively for toxicity assessment, they offer more accurate predictions of drug transport and bioavailability compared to current systems59. These characteristics demonstrate great promise for the in vitro prediction of intestinal toxicity.

Future Liver Toxicity Models In vitro liver models have recently been developed that more accurately mimic the stratified architecture of the liver201-203. These models are the most representative of hepatocyte interactions with the other cell types and the microenvironment. Zhang et al. have utilized 3D printing technique to mimic the vascular interface present in the liver204. 3D printing technology enables tuning the mechanical properties of the matrix, the incorporation of features such as micro-channels, and rapid cell deposition205. While this methodology has not yet been utilized for toxicity testing, it shows promise for future toxicity testing platforms. Kostadinova et al. published a report wherein a NPC fraction and hepatocytes were seeded on opposing sides of a Nylon scaffold to form a tissue201. Hepatocyte function was maintained for 11 weeks, with chemical sensitivity occurring over two weeks. While others have not yet reported toxicity studies using layered models, increases in CYP expression have been shown to occur over a 12-day culture. This model, established by Larkin et al., utilizes a polyelectrolyte multilayer as a Space of Disse mimic to support paracrine heterotypic intercellular interactions and directed signaling203. These cultures allow for each type of NPC to be individually visualized and extracted from culture for further analysis. Most multicellular 3D liver models used for hepatotoxicity studies investigate either cumulative toxicity or hepatocyte-only toxicity. The ability to isolate the response of each cell type to a chemical is a unique feature of

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these 3D liver models. While these cultures have not yet been used to model hepatotoxicity, their stratified architecture, maintenance of CYP expression and ability to isolate each cell type provide a promising platform for studying holistic liver toxicity.

Conclusions There are thousands of chemicals ingested by humans and animals daily at unknown concentrations. The effects of the majority of these xenobiotics on the body are either unknown or poorly understood. Investigation of the toxicity response through in vivo models is resource and time-intensive. Additionally, it is difficult to measure organspecific contributions in animal models. In order to address these deficiencies, engineered tissue models have been developed for toxicity testing. Many of these toxicity models focus on the GI tract and the liver as the majority of chemical absorption and biotransformation occurs in these organs. However, due to the highly metabolic nature of these cell types, these organs rapidly dedifferentiate or lose viability in culture. Efforts have been taken to develop toxicity models that extend cellular phenotypes and maintain biotransformation enzymes. Current models have exhibited some success; although accurate in vitro models have yet to be developed to model first pass metabolism and the corresponding toxicity.

Acknowledgements We gratefully acknowledge financial support from the National Science Foundation (NSF: CBET 1510920). We also acknowledge financial support from the Institute for Critical Technologies and Applied Sciences at Virginia Tech and the Computational Tissue Engineering Graduate Education Program at Virginia Tech.

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For Table of Contents Use Only. In Vitro Intestinal and Liver Models for Toxicity Testing Sophia M. Orbach, Rebekah R. Less, Anjaney Kothari, Padmavathy Rajagopalan

Integration of gut and liver models to recapitulate first pass metabolism of chemicals and xenobiotics.

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Figure 1: GI tract and liver. A) A schematic representation of the GI tract and the liver depicting their orientation in the body B) macroscopic view of the path taken by chemicals and nutrients through enterohepatic circulation C) arrangement of intestinal mucosal cells in crypts and villi D) arrangement of hepatic cells along the liver sinusoid. 183x278mm (300 x 300 DPI)



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Figure 2: Models of GI toxicity. A) An intestinal organoid including the primary cells in the mucosal epithelium B) PCIS showing the maintenance of the layers in the intestinal wall and C) gut-on-a-chip model. 179x274mm (300 x 300 DPI)


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Figure 3: 2D models of liver toxicity. A) Hepatocyte monolayer B) collagen sandwich C) co-culture of hepatocytes with other cell types including NPCs, endothelial cells, fibroblasts, and other epithelial cells 316x63mm (300 x 300 DPI)



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Figure 4: 3D models of liver toxicity. A) PCLS depicting the maintenance of hexagonal liver lobules and the location of the portal triad and central vein B) hepatocyte spheroids in a culture well C) hepatocytes in a bioreactor system. 152x149mm (300 x 300 DPI)


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Figure 5: Integrated GI-liver models of toxicity. A) An integrated microfluidics model incorporating intestinal and liver cells maintaining the in vivo exposure order of the organs to chemicals B) an integrated gut-liver model using PCIS and PCLS. 195x205mm (300 x 300 DPI)


In Vitro Intestinal and Liver Models for Toxicity ... - ACS Publications

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