Towards improved hepatocyte cultures: Progress and limitations

Journal Pre-proof Towards improved hepatocyte cultures: Progress and limitations Marc Ruoß, Massoud Vosough, Alfred Königsrainer, Silvio Nadalin, Silvia Wagner, Sahar Sajadian, Diana Huber, Zahra Heydari, Sabrina Ehnert, Jan G. Hengstler, Andreas K. Nussler PII:

S0278-6915(20)30076-4

DOI:

https://doi.org/10.1016/j.fct.2020.111188

Reference:

FCT 111188

To appear in:

Food and Chemical Toxicology

Received Date: 23 August 2019 Revised Date:

31 January 2020

Accepted Date: 7 February 2020

Please cite this article as: Ruoß, M., Vosough, M., Königsrainer, A., Nadalin, S., Wagner, S., Sajadian, S., Huber, D., Heydari, Z., Ehnert, S., Hengstler, J.G., Nussler, A.K., Towards improved hepatocyte cultures: Progress and limitations, Food and Chemical Toxicology (2020), doi: https://doi.org/10.1016/ j.fct.2020.111188. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

1

Towards improved hepatocyte cultures: Progress and Limitations

2

Marc Ruoß , Massoud Vosough , Alfred Königsrainer , Silvio Nadalin , Silvia Wagner , Sahar Sajadian Diana Huber ,

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Zahra Heydari , Sabrina Ehnert , Jan G. Hengstler

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1) Department of Traumatology, Siegfried Weller Institute, Eberhard Karls University Tübingen, Tübingen, Germany 2) Department of Regenerative Medicine, Cell Science Research Centre, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran 3) Leibniz Research Centre for Working Environment and Human Factors (IfADo), Technical University of Dortmund, Dortmund, Germany 4) Department of General, Visceral and Transplant Surgery, University Hospital Tübingen, Tübingen, Germany

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1

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3,#

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1,#

and Andreas K. Nussler*

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* Correspondence: Andreas Nussler, [email protected] # These authors contributed equally to this manuscript

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Abstract

14

Hepatotoxicity is among the most frequent reasons for drug withdrawal from the market. Therefore, there

15

is an urgent need for reliable predictive in vitro tests, which unfailingly identify hepatotoxic drug

16

candidates, reduce drug development time, expenses and the number of test animals. Currently, human

17

hepatocytes represent the gold standard. However, the use of hepatocytes is challenging since the cells

18

are not constantly available and lose their metabolic activity in culture. To solve these problems many

19

different approaches have been developed in the past decades. The aim of this review is to present these

20

approaches and to discuss the possibilities and limitations as well as future opportunities and directions.

21

Key Words: Primary human hepatocytes, In vitro toxicity testing, 3D culture, Co-culture, 3D micro-tissues,

22

Organ- on-a-chip

23

Introduction

24

Drug development until approval by the U.S. Food and Drug Administration (FDA) may take up to 10

25

years or even longer and the associated costs often amount to hundreds of millions US$ [1]. Moreover, at

26

least 90% of all new substances do not pass the toxicological tests in animal models and clinical phase I

27

to III [2,3]. One of the major challenges of the pharmaceutical industry is the prediction of hepatotoxic

28

properties of a new drug [4]. Today, up to 85% of the cardiovascular and gastrointestinal and over 90% of

29

the hematologic toxicity-associated adverse effects can be predicted in animal experiments [5], which is

30

still the gold standard for preclinical testing [6,7]. However, the predictive power for hepatotoxicity in 1

1

animal experiments is only 50% [3,5]. An important reason is the different expression and activity of drug

2

metabolizing enzymes between human and animals [8]. Therefore, the establishment of in vitro models

3

that reduce the high preclinical and clinical costs and increase patient safety is of high relevance;

4

moreover, the reduction of the number of required laboratory animals is of interest for the regulatory

5

authorities and the scientific community [7,9].

6

Reliable prediction of potential hepatotoxicity is not only crucial in drug approval but equally important

7

during the registration of chemicals, cosmetics and the evaluation of food ingredients & food contaminants

8

[10-12]. Therefore, the development of suitable in vitro models that detect as early as possible adverse

9

effects including hepatotoxicity are important for the risk assessment during the approval procedure of

10

new chemicals. One possible approach would be the the integration of Adverse Outcome Pathways for a

11

specific endpoint in in vitro models [13,14].

12

In the past 40 years, large efforts have been made to establish predictive in vitro test systems. Isolated

13

primary human hepatocytes (PHHs) which can maintain a metabolizing profile similar to the in vivo

14

situation still are the gold standard for in vitro hepatotoxicity tests. However, application of PHHs in routine

15

testing is challenging due to scarce availability and the difficulty to fully maintain their metabolic properties

16

in vitro. Therefore, it has been attempted to replace PHHs by many different cell lines, such as cancer

17

cells or stem cell-derived hepatocyte-like cells, which have advantages and disadvantages compared to

18

PHHs. Moreover, much effort has been invested to improve culture conditions, including 3D cultures,

19

chemical modifiers or fluid-flow cultures which aim to support the metabolic activity of cells [15].

20

Therefore, the purpose of this review is to summarize the state-of-the art of the generation of predictive

21

hepatotoxicity models to highlight advantages and disadvantages of various existing approaches and to

22

address further parameters that still play a role in vivo but are often not taken into account when culturing

23

PHH.

24

1. General aspects of the usage of PHH

25

Liver tissue is mainly available from patients who undergo surgery for liver tumor or metastasis which is

26

only performed in specialized clinics. The treatment strategy of both liver tumors and liver metastases has

27

changed over the years; in the meantime, radiological and other ablative local procedures or systemic 2

1

therapies are often performed before or instead of liver resection. This allows the liver to be operated

2

tissue-sparingly. In view of this approach, less residual tissue is available for research.

3

For the usage of tissue for cell isolation in addition to the donor's consent, the ethical principles

4

summarized in the Declaration of Helsinki, it is advised to follow country-specific regulations to use

5

biomaterials and even more important data protection plus privacy regulations must be taken into account

6

[16]. The isolation process of PHHs as well as the transportation of the cells to a laboratory is associated

7

with loss of viability, altered metabolism and gene expression. Thus, to overcome these shortcomings and

8

to improve the isolation and transport of PHHs different approaches have been developed in recent years.

9

Some include techniques, where PHHs do not have to be isolated from the tissue, or can be used without

10

plating as suspension culture. The advantages and disadvantages of these approaches as well as

11

different methods of PHH isolation, storage and shipment are discussed in the following section.

12

1.1 PHH isolation, purification, storage and shipment

13

PHHs can be isolated from normal liver tissue that is resected together with primary or secondary liver

14

tumors [17], or from whole livers or individual liver segments not eligible for transplantation, for example

15

because of excessive steatosis, abnormal anatomy, traumatic injury or lack of a suitable recipient [18]. It is

16

possible to isolate PHHs from these livers by applying mechanical, chemical and enzymatic methods, or

17

by a combination of these procedures [19]. Since it is difficult to achieve sufficient viability through

18

mechanical or chemical methods, the isolation of the cells is preferrably done via an enzymatic method,

19

known as two-step collagenase digestion [17,19]. The viability of the cell suspension can be enhanced by

20

Percoll density centrifugation [20]. Then, purified cells can either be directly plated onto different matrixes

21

or cryopreserved according to the corresponding protocols. Shipment or storage on ice in adequate cold

22

storage solutions has also been described elsewhere [15].

23

Although numerous protocols for PHH isolation and shipment are available, several factors usually limit

24

the quality of the obtained cells (Table 1) [21,22].

25

Table 1 Challenges and limitations of PHHs from donors, their isolation and shipment

Donor/ tissue

3

- Most donors may be of older age and have usually a disease history: no healthy tissue - Donors are often pre-medicated, which may result in damaged PHHs and/or altered metabolic properties of PHHs

- Often, only a small liver tissue specimen is available for PHH isolation Isolation process

Storage/shipment

- The isolation of PHHs must be carried out as fast as possible, otherwise viability will decrease - Enzymatic digestion, applied for the isolation of PHHs, may lead to irreversible metabolic changes - The viability of the PHHs can be improved by a Percoll density centrifugation, which is, however, associated with a loss of living cells, especially if much fat is stored in the PHH - PHHs can be shipped overnight in a suspension on ice, which is unfortunately associated with a loss of PHHs - The shipment of plated PHHs at 37°C reduces the loss of viable cells, but is logistically more complicated - PHHs can be cryopreserved. However, the viability and metabolic properties after thawing are strongly dependent on the initial PHH quality before freezing and the medical history of the donor

1 2

When PHHs are isolated from human liver resections, it is difficult to isolate large numbers of high quality

3

cells. As depicted in Figure 1, representing the data from our patients, many donors are older than 50

4

years, frequently with severe pre-existing liver diseases, and/or a chemotherapeutic pretreatment, all

5

leading to low-quality PHHs.

6 Age of the liver donor

Diagnosis of liver donor 40 15%

35

9%

6% 6% 43%

3% 3%

Proportion of the age group in total donor number in %

15%

30 25 20 15 10 5

7

HCC Haemangioma Cholestasis Metastases of colon carcinoma

Cholangiocarcinoma Focal nodular hyperplasia Biliary cirrhosis Metastases of other tumors

0

>30 <30 30-39 40-49 50-59 60-69 70-79 Donor Age in years

≥80

8

Figure 1 Summary of age and diagnosis of patients undergoing liver resections used for PHH isolation

9

Pre-existing diseases as well as their therapy can affect both the metabolic properties of PHHs and their

10

viability [23-25]. Residual tissue from tumor surgery often is of such a small size that only a low number of

11

cells can be yielded. As shown in one study, 13 ± 11 x10 living PHHs per g liver tissue can be isolated

12

[26] however, this is mostly not achievable from diseased livers. 4

6

1

Additionally, the cells should be isolated as quickly as possible after the removal of the tissue, otherwise

2

they rapidly lose their viability. For example, it has been shown that after a period of 3-5 hours between

3

hepatectomy and the start of the perfusion procedure, the viability of the viability of the cells decreases by

4

up to 50% compared to the cells which were isolated within first three hours after resection [23]. Moreover,

5

it has been shown, that the cell isolation process causes metabolic changes in isolated PHHs, which

6

cannot be reversed during the following cultivation [27]. As shown in a study, these metabolic changes are

7

not irreversible, at least in the rats. After transplanting the rat hepatocytes back into an animal, these cells

8

were able to restore their differentiated phenotype, whether the functionality of the cells can be fully

9

restored and whether this process is also possible in human cells could not be shown yet [28].

10

Metabolic changes caused by the isolation process has multifarious causes. The digestion of the liver

11

tissue results in the release of proteases and pro-inflammatory cytokines, that can lead to inflammatory

12

responses in the PHH . In addition, collagenase digestion leads to the destruction of cell-cell and cell-

13

matrix contacts, that are essential for the functionality of PHH [29]. The interruption of the blood supply

14

and the subsequent re-perfusion, leads to ROS and hypoxia-related damage to cells [29,30]. In response

15

to these stress factors, significant changes in the hepatocyte gene expression occur, that are intended to

16

ensure the survival of the cells and for adaptation to the new conditions [29]. In the past, various

17

approaches have been tested to reduce possible damage caused by the isolation process. In addition to

18

the inhibition of apoptosis as well as the inhibition of Kupffer cells to release reactive oxigene

19

intermediates the addition of N-acetylcysteine to the isolation medium are suitable approaches to improve

20

cell quality [15,29-31].

21

The shipment and the storage of PHHs is also associated with a loss of cell viability and/or reduced cell

22

function [15]. Although PHHs can be cryopreserved, the quality of the cells after thawing strongly depends

23

on the initial quality of the cells (viability, fat content, storage of the liver before isolation) and additional

24

factors from the medical history of the patient (drug intake, smoking, diabetes, obesity). These factors

25

affect not only the quality of cryopreserved PHHs but also the metabolic activity of the isolated cells after

26

thawing [15]. These changes are partly an effect due to damage to the hepatocytes mitochondria, which

27

are very sensitive to freezing and thawing. The thawing process results in an impairment of complex 1

28

activity, which leads to low intracellular ATP concentrations. In addition, the release of cytochrome c by 5

1

the damaged mitochondria leads to an induction of apoptosis [32]. However, cryopreservation is still an

2

essential technique to use PHHs since this approach is currently the only technique that allows cell

3

storage and transport[15]. Moreover, it allows to schedule cell culture experiments in advance.

4

The transport of the cells in suspension on ice is another possible alternative, but the same problems in

5

terms of reduction in viability and altered metabolic properties may occur [33]. Overnight shipment of cells

6

plated on a scaffold or in 2D at 37°C is another possibility. According to our data, shipment of plated cells

7

can reduce the loss of cells and increase their metabolic activity compared to shipment in suspension [34].

8

However, this method of shipment is logistically rather complex, since an adequate temperature should be

9

maintained during the whole transport period [34].

10

As described above, many factors have an influence on the viability of the PHHs and their metabolic

11

activity. Therefore an extensive characterization of the PHHs before their use is required. In one work

12

published by Vinken and Hengstler various quality criteria were described. These include, in addition to a

13

viability of at least 90% and a polarized and hexogenous morphology. Additionally, the expression and

14

activity of various functional markers, such as different phase I / II enzymes should be checked. For

15

toxicity studies, it is also useful to test known hepatotoxins such as paracetamol as a positive control for

16

the endpoint of interest [35]. However, as different studies show, the viability after isolation and transport

17

is often less than 90% [26,34,36]. When using such cells, it is, therefore, to be expected that the low

18

viability harms the functionality of the cells. This is undoubtedly one reason why, for example, in the EMA

19

guideline CPMP / EWP / 560/95 a viability of >80% is required for the investigation of drug interactions

20

[37]

21

1.2. Liver slices as a possible alternative?

22

Changes in PHH metabolism induced by the isolation and purification procedure are inevitable. Moreover,

23

tissue dissociation destroys liver architecture which also negatively influences the metabolism. So far, the

24

only known alternative method, where cells do not need to be isolated and can be kept in their natural

25

cellular environment, is the use of so-called liver slices, where the zone-specific activity of the

26

Cytochrom P450 (CYP) enzymes is partially maintained [38]. In terms of xenobiotic metabolism this

27

technique allows mimicking the in vivo situation, however, only for a short time period [11]. The crucial

28

disadvantage of this cultivation form is that the tissue is disconnected from the blood stream and therefore 6

1

no longer sufficiently supplied by nutrients. Consequently, cultivation of liver slices over longer time

2

periods leads usually to necrosis within 24 to 72 h, accompanied by a reduction of enzymes responsible

3

for drug metabolism [11]. To maintain the viability of the liver slices over a more extended period, thin liver

4

slices are necessary, which enables that all cells within the sliced can be supplied with sufficient oxygen

5

and nutrients [39]. As shown in the current study, it is possible to produce liver slices with a thickness of

6

250 µm, using a precision cutting process, which can be cultivated for up to 15 days. The perfusion takes

7

place here passively using a rocking platform [40]. An improvement in the perfusion of the liver slices by

8

the development of an active perfusion technique as well as an adaptation of the medium, which takes

9

into account the requirements of all cells located in the liver could be helpful to further increase the

10

maintenance of the viability and function in the future [41-43].

11

1.3 PHH suspension culture

12

Besides the above-described method with liver slices, PHHs in suspension culture can be used for short

13

term experiments. Compared to the metabolic changes in the cells, which are immediately plated onto

14

culture plates after the isolation, the metabolic changes due to cooling or freezing are significantly lower

15

[44]. In the same line of evidence, it has been shown that cells can be shipped on ice or cryopreserved for

16

further use in suspension culture. Since PHHs in suspension culture express specific liver functions at a

17

physiological level, they may represent the most suitable system to generate metabolites from test

18

compounds. Because PHH cells show a high metabolic activity in suspension culture, this is additional

19

optimally suited as a reference to estimate the functionality of the cells during the later culture. Literature

20

data from suspension cultures would be helpful as reference to compare the measured values. We have

21

summarized some reference values for the most important phase I/II enzymes as well as for the

22

production of urea in Table 2. However, it becomes rapidly clear that there is no consistency in the

23

existing literature regarding the used substrates, the substrate concentrations, and the method chosen for

24

normalization, which makes it extremely difficult to compare the existing data which each other [36,45]The

25

main disadvantage of this cultivation technique is that PHH suspension cultures are only stable for a few

26

hours, which makes long-term experiments impossible yet [46]. Because of this limitation, suspension

27

cultures are not suitable for the investigation of hepatotoxicity. Although suspension cultures can form

28

relevant reactive metabolites, the resulting hepatotoxicity step is often too slow [11] to be visualized due to

29

its short stability. 7

1

Table 2 Functional criteria of the usage of PHHs for metabolism and toxicity studies

Responsible metabolic

Used Substrates

enzyme CYP1A2 CYP2B6

Conc. (µM)

Analyzed metabolites

Reference

Phenacetin

10

Acetaminophen

7.04 1

[36]

Tacrine

1

Hydroxytacrine

1.51 2

[45]

Bupropion

1

Hydroxybupropion

10 CYP2C9

Analyt concentration

Diclofenac

0.7

2

106.9

1

[45] [36]

4-Hydroxydiclofenac 1

3.1 2

[45]

S-mephenytoin

50

4-Hydroxymephenytoin

3.94 1

[36]

Bufuralol

10

1-Hydroxybufuralol

4.92 1

[36]

Dextromethorphan

1

Dextrophan

9.6 1

[45]

CYP 2E1

Chlorzoxazone

50

33.1 1

[36]

17.5 1

[36]

CYP3A4

Midazolam

CYP2C19 Phase I CYP2D6

6-Hydroxy chlorzoxazone

5 1′-Hydroxymidazolam 1

32.5 7-Hydroxycoumarin

Phase II

UGT/SULT

7Hydroxycoumarin

glucuronide 1

7-Hydroxycoumarinsulfate

Urea

NH4Cl

10 mM

Urea

1

[45]

37.6 1

[36]

3.3 1

[45]

2.53 1

[36]

Production

2

1) pmol/min/106 cells 2) pmol/min/mg protein 3) mol/min/106 cells

3

2. Techniques and approaches to improve the metabolic activity of cultured PHHs

4

Despite the above stated disadvantages, PHHs plated in conventional 2D cultures are still the "gold

5

standard" for long term in vitro metabolism and toxicity testing. Another difficulty is that despite the high

6

regenerative potential of the liver in vivo, no significant cell proliferation can be observed after cell isolation

7

[15]. Presently, it is difficult to provide freshly isolated PHHs for in vitro testing in sufficient numbers.

8

Consequently, in the past decades many different approaches have been developed and tested with the

9

aim to maintain the metabolic activity of cells for extended periods. As shown in Figure 2, the goal of many

10

research strategies is to mimic the in vivo environment as close as possible to provide cells with the

11

optimal culture conditions. 8

1 2

Figure 2 Techniques: approaches and ideas towards improved PHH cultures with a maximal metabolic

3

capacity and an adaptation towards in vivo conditions

4 5

2.1 Optimization of culture media for PHH cultures

6

For the cultivation of PHHs many researchers use Williams E (WE) or DMEM medium, supplemented with

7

antibiotics, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), sodium pyruvate, glutamine as

8

well as nonessential amino acids, Fetal calf serum (FCS), hydrocortisone and insulin [17]. Several studies

9

have investigated whether the addition of further culture supplements can increase the metabolic activity

10

of PHHs. One study has shown that the addition of Hepatocyte Growth Factor and Oncostatin M (OSM)

11

improved the metabolism of the cells [47]. Others have shown that the addition of antioxidants, such as 9

1

vitamin C, to the culture medium compensates oxidative stress caused by the cultivation or isolation

2

process, and improves the metabolic capacity of PHHs [48].

3

The use of FCS in culture medium may lead to difficulties for standardization. Since FCS is an animal

4

product, its exact composition may vary among different lots and different producers due to seasonal and

5

geographical differences [49]. Therefore, many laboratories reserve large lots from a single provider if the

6

tested lot is suitable.

7

It has been shown that PHHs can also be cultured in serum-free media for up to 6 days without losing the

8

metabolic capacity [50]. However, this particular study lacks a direct comparison to FCS-containing

9

medium, which makes it difficult to interpret these results. Nevertheless, the advantage of this approach is

10

the fulfillment of the 3R principle replacement [51] and the possibility of future standardization.

11

2.2 Stabilization of the hepatic phenotype – by molecular biological or pharmaceutical

12

approaches

13

Another approach to maintain the metabolic capacity of PHHs includes epigenetic changes,

14

pharmacological interventions in cell signaling or transfection of the PHHs with genes that support

15

maintenance of the mature hepatic phenotype. The aim of these approaches is to delay de-differentiation

16

of PHHs or stimulate them to proliferate.

17 18

2.2.1 Stabilization of the hepatic phenotype by epigenetic modifications

19

Epigenetic changes contribute to the rapid loss of PHH function in vitro [52,53]. Epigenetic modifications,

20

including DNA and histone modifications, play an essential role in the organization of the chromatin

21

structure and, consequently, in the control of gene expression [53]. The inhibition of DNA methylation by

22

treatment with, e.g.; 5-azacytidine (5-AZA) and inhibition of histone deacetylation by treatment with, e.g.;

23

Trichostatin A (TSA), results in an altered expression of the epithelial marker gene E-cadherin as well as

24

genes involved in proliferation and drug metabolism in hepatoma cell lines [54-56]. Therefore, epigenetic

25

programming may play an essential role in maintaining a differentiated phenotype in PHHs [57-59]. We

26

measured epigenetic changes during PHH cultures with and without stimulation with the epigenetic 10

2

1

modifiers 5-AZA and vitamin C by using an Epigenetic Chromatin Modification Enzymes RT Profiler PCR

2

Array (Figure 3). Different Epigenetic Chromatin Modification Enzymes such as SET Domain Bifurcated

3

Histone Lysine Methyltransferase 2 (SETDB2), Aurora kinase A (AURKA), establishment of sister

4

chromatid cohesion N-Acetyltransferase 2 (ESCO2) and Cysteine-rich protein 2-binding protein

5

(CSRP2BP) were de-regulated during cultivation. De-regulation was even stronger in different hepatic cell

6

lines, suggesting that these enzymes play a crucial role in hepatic differentiation. The treatment with 5-

7

AZA and vitamin C shows sporadic positive effects on the stabilization of the expression profile e.g.; in

8

AURORA and SETDB2, but on day 7 a general downregulation of most of the measured genes is

9

observed, which suggests that this kind of treatment does not lead to a preservation of the genetic

10

phenotype of PHHs in long term culture. However, other studies had shown a positive influence of the

11

epigenetic modification on the expression of genes that influence hepatic differentiation like HNF4α or on

12

the expression of drug-metabolizing enzymes in PHHs. These positive effects could be achieved by the

13

usage of 5-AZA, but also by the use of various HDAC inhibitors including TSA [53]. Although the de-

14

differentiation was slowed down as shown in these studies by the epigenetic modification of the cells but

15

not stopped [57-59]. Another limitation of this approach is the lack of specificity of the epigenetically active

16

substances used. The treatment with epigenetic modifying substances leads to a general increase in gene

17

expression, which is not limited to hepatospecific genes. For example, it has been shown in one study that

18

after treatment of HepG2 cells with 5-AZA also genes of spermatogenesis are expressed; this result

19

shows one important limitation of this approach [60].

20

11

1 2

Figure 3 Epigenetic changes during primary human hepatocyte (PHH) culture quantified by the Epigenetic

3

Chromatin Modification Enzymes RT Profiler PCR Array; partially from [61]

2

4 5 6

12

1

2.2.2 Stabilization of the hepatic phenotype by pharmaceutical intervention

2

The loss of the differentiated PHHs phenotype during culture is associated with the so-called epithelial-

3

mesenchymal transition (EMT). Therefore, investigators tried many approaches to stabilize the phenotype

4

by using the Extracellular Signal-Regulated Kinase (ERK) inhibitor PD0325901, the GSK-3β inhibitor

5

CHIR99021, or a combination of both. The authors conclude that the ERK 1/2 as well as the GSK-3β

6

pathways are involved in the maintenance of the epithelial phenotype of PHHs [62].

7

2.2.3 Transfection approaches to facilitate PHH proliferation or maintain their metabolic activity

8

As described above, insufficient availability of PHHs is a major challenge. The injured liver is capable to

9

fully regenerate itself after losing 68 ±10% of its original organ mass through the proliferation of the

10

remaining cells [63]. In contrast, in vitro expansion of PHHs still remains challenging [64]. Therefore,

11

several approaches have been developed to induce their proliferation. In one of them, PHHs were

12

transduced with virus particles, containing human papilloma virus (HPV) E6 and E7 genes. To stabilize the

13

epithelial phenotype of PHH, they were incubated with the Mitogen-activated protein kinase (MEK) 1/2

14

inhibitor U0126 to inhibit the ERK 1/2 pathway. Since high expression of E6 and E7 results in PHH

15

immortalization and loss of differentiation, colonies with low HPV expression of E6 and E7 were selected.

16

Expression of E6 and E7 upregulates the OSM receptor. Under these conditions, OSM stimulation results

17

in the proliferation of the PHHs. The removal of OSM terminates proliferation followed by differentiation

18

into metabolically active PHHs, which express CYP enzymes such as CYP 2C9 or CYP 2D6 comparable

19

to PHHs. However, the expression of CYP 3A4, which is essential for the metabolism of many drugs, is

20

weak compared to PHH [65]. In another study, PHHs were immortalized by transduction with a subunit of

21

human telomerase reverse transcriptase (hTERT). Since hTERT transduced PHH showed a reduction of

22

hepatocyte-specific functions in long-term culture [66], PHHs were transiently transfected with Hepatocyte

23

nuclear factor 4 alpha (HNF4α), a transcription factor that supports many differentiated functions of PHH

24

and their epithelial phenotype. HNF4α expression enhanced the mRNA levels of CYP 3A4 and albumin as

25

well as the detoxification capacity of ammonia [67]. In general, several approaches to ameliorate the de-

26

differentiation process of PHH or to generate cells, which can proliferate and establish some functions of

27

PHH were tested in recent years, however, a model that provides an unlimited availability of cells and fully

28

reflects the metabolism of PHHs, cannot be generated by any of these techniques so far. 13

1

2.2.4 Reprogramming of mature PHHs into expandable bipotent progenitors using chemical

2

induction

3

Some studies induced the proliferation of mature mouse hepatocytes by chemical induction with a cocktail

4

of small molecules (Y-27632, A-83-01, and CHIR99021). The authors claim that it is possible to

5

differentiate these cells into mature hepatocytes and biliary epithelial cells. As shown by albumin secretion

6

and the production of urea, the modified cells can achieve a certain degree of differentiation, but do not

7

have the same properties as freshly isolated mouse hepatocytes [68,69]. In summary, this is an interesting

8

approach, which requires further optimization to improve the metabolic function of the cells.

9

2.3 Co-culture of PHHs and non-parenchymal cells

10

Hepatocytes make up about 80% of all liver cells and are responsible for most hepatic functions [70].

11

Other cell types of the liver, the non-parenchymal cells (NPCs), particularly sinusoidal endothelial cells,

12

Kupffer cells and hepatic stellate cells play a crucial role in the development and progress of several

13

diseases, such as stellate cells in liver fibrosis and Kupffer cells in lipopolysaccharides (LPS) mediated

14

hepatotoxicity [71,72]. Co-cultures with one of these cell types or a mix of different NPCs help to maintain

15

the function of PHHs via exchange of metabolites and cytokines [73]. Whereas co-culture with endothelial

16

cells and fibroblasts focuses on increasing the function of PHHs the co-culture of PHHs with Kupffer cells

17

and stellar cells rather are used to mimic pathological mechanisms in vitro. For example, a co-cultures of

18

PHHs with immune cells can be used to study immune-mediated drug-induced hepatotoxicity [72].

19

Moreover, co-cultures of rat hepatocytes with stellate cells or their lipid extract, allows to study interactions

20

between these cell types. Interactions of co-cultivated cells occur by the release of cytokines and

21

metabolites [74]. Nevertheless, the challenge remains that NPCs tend to change their phenotype due to

22

the isolation and cultivation process, e.g. stellate cells and Kupffer cells may be activated in culture and

23

sinusoidal endothelial cells tend to lose their fenestration [15,75].

24

2.3.1 Maintaining the hepatic functions by using co-cultures

25

PHH functions have been stabilized to some degree by the co-cultivation of PHHs with liver sinusoidal

26

endothelial cells (LSECs) or non-liver endothelial cells (e.g. HUVEC). It was shown that LSECs, in

27

contrast to human umbilical vein endothelial cells, were able to stabilize albumin production throughout a

14

1

cultivation period of 11 days. Moreover, experiments with 3T3-J2 fibroblasts plus endothelial cells and

2

PHH maintained metabolic properties for up to three weeks [76].

3

2.3.2 Identification of Immune-mediated drug-induced hepatotoxicity by co-culture with immune

4

cells

5

Analysis of immune-mediated drug-induced hepatotoxicity is limited in conventional PHH monocultures,

6

since the mode of action requires interaction with immune cells in the presence of drugs. One model to

7

investigate immune-related responses to drug exposure is the co-culture of rat hepatocytes with Kupffer

8

cells. In this model it was shown that test compounds known to cause immune-mediated as well as direct

9

hepatotoxic effects resulted in enhanced or mitigated hepatocellular injury under co-culture conditions

10

[77].

11

2.4 Optimization of oxygen tension

12

In conventional cell cultures, the CO2 concentration in the incubator is usually adjusted to 5% CO2 and a

13

pH of 7.4 for stabilization of the pH of the buffered culture media [78]. It seems plausible to also adapt

14

oxygen tension to levels similar to the in vivo situation. The partial pressure of oxygen in the liver is 40.6

15

mmHg [79], which is much lower compared to the generally applied cell culture conditions which is 150

16

mmHg [62]. Indeed, recent studies support a role for oxygen tension and lobular zonation [80]. In the

17

same line evidence, it has been demonstrated that oxygen tension has an effect on the metabolic

18

characteristics of rat hepatocytes already in the first 9 hours of cultivation [81]. Various other studies have

19

shown that the application of in vivo like oxygen concentrations not only delays the de-differentiation

20

process of rat hepatocytes, but also improves the function of hepatic cell lines [62,82]. Thus, the use of

21

physiological oxygen concentrations could reduce the generation of ROS, that are associated with DNA

22

damage and stabilizes the functions of the rat hepatocytes [62].

23

2.5 Physiological topology of hepatocytes

24

The function of individual hepatocytes in a single liver lobule is regulated by different gradients. In addition

25

to a gradually decreasing oxygen concentration from the periportal to perivenous zone of the liver lobe,

26

the glucose concentration as well as the concentrations of various hormones are also decreases from the

27

periportal to perivenous zone [83].

15

1

Together with a gradual expression of the Wnt expression, which is highly expressed in perivenous

2

hepatocytes, these lead to a functional zonation of the hepatocytes within the liver lobe. Various studies

3

have attempted to imitate the hepatic zonation in vitro. For example, in one study, a model was developed

4

which allows to mimick the zone-specific hepatotoxicity of acetaminophen. This could be reached by a

5

gradual induction of wnt signaling within a hydrogel gel containing channel using the GSK3β inhibitor

6

CHIR99021 [84]. In another study, it was possible to generate periportal and perivenous PHHs by adding

7

hormones such as insulin and glucagon, which correspond in their function to PHHs of the periportal or

8

perivenous zone. Hormone stimulation led in this study to a three times faster gluconeogenesis in

9

periportal hepatocytes and eight times faster glycolysis in perivenous hepatocytes [85]. Although these

10

studies show that different factors influence the functional zonation of the liver, an in vitro model that

11

combines all these factors is necessary to develop for mimicking the in vivo situation.

12

2.6 Fluid-Flow cultures

13

PHHs are supplied with nutrients and exposed to endogenous metabolites and xenobiotics through the

14

bloodstream within the sinusoids [80]. A side effect of this blood supply is continuous shear stress caused

15

by the blood stream. Compared to endothelial cells, which are exposed to shear stress of 1 to 30 dyne/

16

cm in vivo, a shear stress of 0.1- 0.5 dyne/cm has been reported to be optimal for the cultivation of

17

PHHs in vitro [86,87]. This low shear stress seems to be support maintenance of hepatic functions [86].

18

One of the first studies, which tested the effect of a fluid-flow culture on the maintenance of PHHs

19

metabolic activity showed a positive effect of the fluid-flow system on the expression and metabolic activity

20

of some drug metabolizing enzymes (e.g.; CYP 3A4, CYP 2C9, and UGT2B4/7). It is interesting to note

21

that some hepatic functions, like the production of albumin, the detoxification of ammonia as well as the

22

gene expression and activity of CYP 2D6 are apparently not influenced by fluid-flow cultures [86].

23

In a different setup, it has been shown that the metabolic activity of the enzymes CYP 1A2 and CYP 3A4

24

in PHHs cultured in a fluid-flow system could be maintained on a constant level between day 3 and day 10

25

without FCS supplementation. The enzyme activities of CYP 2C9, CYP 2D6 and CYP 2B6 were reduced

26

at day 10, compared to day 3 in both FCS-free and FCS-supplemented culture [88]. Recently, Lonza

27

(Basel, Switzerland) has launched the Quasi Vivo QV900 Flow System to culture PHHs in a fluid-flow

28

culture setup. As described before for other fluid-flow cultures, this system showed no improved albumin

2

16

2

1

production compared to static cultures. While no improved albumin production was observed in this setup,

2

the enzyme activities of CYP 3A4, CYP 2B6 and CYP 1A2 were significantly increased over 21 days in

3

comparison to static cultures [89]. In summary, these results indicate that fluid-flow cultures may improve

4

the maintenance of some PHH functions, such as the activity of the CYP enzymes. Unfortunately, there is

5

no direct comparison to day 0, as well as in many other studies; this information would be valuable, since

6

the loss of differentiated functions of PHHs is most pronounced in the first days of cultivation and is later

7

stabilized at a lower level. For example, the mRNA expression of CYP 3A4 is already reduced by 70-80%

8

after 4-6 hours in culture, which shows how quickly the de-differentiation process takes place [90]. In

9

addition, the comparison of different systems is often difficult, as methods used in 2D cannot be easily

10

translated into a fluid-flow system. Therefore, preference is given to systems in which the fluid flow

11

cultured cells can be transferred to a conventional plate for measurement. For example, this can be

12

reached as shown by Buesch et al. by the culture of the cells under fluid flow conditions on cover slips and

13

transferring them for the measurement to a conventional well plate [89]. Alternatively, it is also possible to

14

harvest cells that were cultured under different conditions to compare mRNA and protein levels of the

15

cells.

16

2.7 Influence of different matrixes on PHH cultivation and adherence

17

Usually PHHs are plated onto collagen-coated plates. The collagen used for coating can either be isolated

18

from rat tails or synthetically produced [91]. Collagen-coating allows the adherence of PHHs, but does not

19

completely represent the natural extracellular matrix (ECM) of the liver, that consists of collagen type I in

20

addition to fibronectin and to a lesser extent collagen type III, IV, V and VI [92]. As an alternative to rat tail

21

or commercially available products, some researchers apply Matrigel, that contains various ECM proteins

22

and growth factors extracted from Engelbreth-Holm-Swarm mouse sarcoma [93]. Although Matrigel is

23

frequently its drawbacks are obvious: it does not reflect the natural ECM of the human liver, and its exact

24

composition is unknown and varies between different production lots [94]. Moreover, it is an animal

25

product and it is unknown what it does to human cells and should be therefore minimized in human

26

toxicological experiments. As an alternative, recombinant laminin or ECM protein fragments could be

27

applied as coating material for the cultivation of PHHs [95,96]. ECM proteins, such as fibronectin or

28

collagen VI, as suitable matrixes for PHH cultures as shown by the ECM Select Array (Fig. 4). By applying

29

fibronectin, cell adherence can show a 1.7-fold increase compared to the commonly used collagen coating 17

1

suggesting that a combination of different ECM proteins may even improve attachment rate and function

2

of PHHs too. Indeed, this was shown in an earlier work, where plates were coated with a combination of

3

fibronectin and collagen I [97]. Another approach is the use of synthetic cell adhesion peptides that may

4

mimic ECM changes triggered by pathological processes, such as in liver fibrosis [98].

5 6

Figure 4 Results of the ECM Select® Array Kit Ultra-36 (Advanced Biomatrix, San Diego, USA) performed

7

with primary human hepatocytes (PHHs); 250,000 cells were plated in a total volume of 5 ml on the Array.

8

The Array is a microscope glass slide functionalized with a hydrogel on the surface. 36 ECM conditions

9

are deposited onto this hydrogel surface. Microscopic pictures of all conditions were taken 12 h after

10

plating, PHHs attached on the surface were counted in all conditions and normalized to the condition with

11

collagen I, which is usually used for PHH cultivation. The green color shows a higher attachment

12

compared to Collagen Type I coating whereas the red color shows a lower attachment compared a

13

coating with Collagen Type I. ECM combinations that were not included in the array are shown as white

14

boxes.

15

2.8 Effect of the substrate stiffness on the metabolic activity of PHH

16

The matrix stiffness is an important mediator of cell behavior, which regulates cell signaling and has an

17

impact on many cellular processes, such as cell proliferation and cell differentiation [99,100]. With a 18

9

1

stiffness of >10 Pa, the stiffness of cell culture plastic is significantly different than the elasticity of human

2

tissues [101]. Various liver diseases, such as liver fibrosis [102], are associated with altered liver stiffness.

3

Since changes in the liver rigidity influence metabolic activities [103], stiffness should be considered as an

4

important parameter in an in vitro culture model. Values of the healthy liver given in literature vary

5

between 150 Pa and 6 kPa [102,104]. This large range results from the different methods used in the

6

clinic as well as in experimental research. While the value of 150 Pa refers to the invasive measurement

7

of liver tissue stiffness by Atomic force microscopy (AFM), 6 kPa often is reported from non-invasive

8

FibroScan® measurement used in the clinic [102,104]. Since cells are embedded in the ECM and not

9

adhere to the surface of the liver capsule, the value of the invasive measurement seems more relevant for

10

the development of PHH cell culture system. However, this kind of measurement has also its limitations

11

since during sample preparation, which is necessary in case of invasive stiffness measurement of the

12

liver, the tissue must be frozen, which could result in changes in the nature of the tissue and therefore also

13

the rigidity. In vitro culture of PHHs seeded on a collagen matrix with an adjusted stiffness showed that the

14

matrix stiffness has effects on the morphology and the function of mouse hepatocytes [104]. In a relatively

15

narrow range around the normal liver stiffness of 150 Pa cells showed the highest metabolic function,

16

while a matrix stiffness of liver fibrosis had a strong negative effect on cytoskeletal tension (e.g. active

17

forces developed by the cell) [105] resulting in significantly reduced hepatocyte-specific functions [104].

18

The effect of liver stiffness on the expression of various CYPs and drug transporters has also been

19

demonstrated in the clinic. In one study, the drug metabolizing capacity of chronic alcoholics was

20

examined. Patients with a liver stiffness <8 kPa showed an induction of the CYPs and drug transporters,

21

whereas the group with a stiffness over 8 kPa (fibrotic tissue) showed an inhibition in the CYP enzymes

22

[103]. However, it should also be considered that liver fibrosis is a complex process, associated with

23

infiltration of inflammatory cells and often with bile salt overloading the liver tissue, which may also have

24

an influence on the metabolic activity of the cells [106].

25

3. Cultivation of liver cells on three-dimensional systems

26

PHHs in the liver lobules are arranged towards a polarized epithelium, that is essential for the

27

sophisticated functionality of the liver [107]. To achieve this, cells are connected to ECM at the basolateral

28

or blood side and form bile canaliculi at the apical side. At their interface, hepatocytes form tight, 19

1

anchoring and gap junctions [108]. Cell junctions as well as the establishment of polarization which

2

includes an apical (luminal) and basolateral side of each cell, play essential roles in the performance of

3

liver-specific functions [108]. For the cultivation of functional PHHs, it is therefore mandatory to enable

4

cells to polarize through the formation of cell-cell junctions as well as to build connections to the

5

surrounding matrix [15,108]. Since the ECM and the tissue stiffness can only be rudimentary mimicked in

6

a 2D monoculture, the possibility to generate cell-matrix contacts in 2D models is very limited. In 3D

7

cultivation techniques cells are embedded into a matrix, which supports establishment of polarity.

8

3.1 Collagen-sandwich cultures and hydrogels

9

The most frequently used 3D culture is the cultivation of cells in a hydrogel or in a sandwich consisting of

10

(natural or synthetic produced) ECM proteins, such as collagen or Matrigel. These 3D culture systems

11

recapitulate the sheets of hepatocytes in the liver, whereby the culture medium side represents the

12

basolateral blood side and the cell-cell interface the apical side. PHHs are usually suspended in the gel

13

and directly plated onto culture dishes. Alternatively a so-called sandwich culture can be used, in which

14

the cells are plated onto a thin layer of the matrix and after adherence, covered with a second layer of the

15

same matrix [109]. In contrast to the 2D culture, the cells in the sandwich culture have contact to the ECM

16

on two sides, which supports the development of polarity and expression of drug transporters. In the 3D

17

sandwich culture, some substances can be better taken up into the cell. Also the metabolic capacity may

18

be better maintained [110]. As described before also a low surface stiffness could increase the metabolic

19

activity of the hepatocytes. This could be another reason for the increased metabolic activity of cells

20

cultured in a collagen gel, whose stiffness lies between ~80-200 Pa, and is therefore similar to the in vivo

21

situation [111]. The usage of hepatocytes in hydrogels has its disadvantages as well. Similar to a collagen

22

or Matrigel, this set up does not correspond to the natural ECM. The significant problem is that the cells

23

are trapped in the matrix. Dead cells cannot be washed out and therefore may affect other cells. The

24

thickness of the gel on top of the cells may delay the diffusion of nutrients and the removal of waste

25

products.

26

3.2 Culture of PHHs on scaffolds

27

PHHs can also be cultured on scaffolds [109], where they grow within pores. This may allow removal of

28

dead cells and a better supply with nutrients but this is difficult to verify [77]. A broad range of materials as 20

1

well as diverse manufacturing processes are available that were applied in the development of scaffolds in

2

recent years [112]. The properties of the scaffold should reflect the corresponding in vivo conditions. In

3

addition to the already described criteria, such as ECM requirement and rigidity, the morphological

4

properties such as porosity and pore size, as well as physicochemical properties, such as permeability

5

and the interconnectivity of the pores, are essential for a scaffold [34,113]. As seen in the scanning

6

electron microscope (SEM) image of a healthy human liver (Figure 5), PHHs are embedded in a porous

7

ECM structure. The pore size corresponds to 20-30 µm which is similar to the diameter of PHHs [43]. In

8

vivo, the individual ECM-fibers such as elastin and collagen form a much finer ECM mesh [114]. It could

9

be shown in in vitro that the size of the pores has an important impact on the activity of the PHHs. Small

10

pores of around 10 µm helps to maintain the morphology and thus the hepatic function, although the cells

11

can not completely penetrate the pore because of their size. In contrast, larger pores of 80 µm may also

12

be useful for the cultivation of PHHs, since several cells in a pore can form cell-cell contacts in addition to

13

interacting with the matrix [113,115].

14 15

Figure 5 Scanning electron microscopy (SEM)- Image of the porous structure of a healthy liver tissue

16

To establish a static 3D culture with properties similar to the in vivo situation, it should be ensured that the

17

cells are able to penetrate the scaffold after seeding and that they are sufficiently supplied with nutrients

18

during the cultivation period. Since the supply cannot be provided through the bloodstream as in vivo but

19

only by passive diffusion, the pores need to exceed critical sizes. Scaffolds with pore size between 80 and

20

100 µm have been reported to be adequate for cultivation of PHHs in various studies [34,115-117]. 21

1

Another important precondition to enable a sufficient nutrient supply is a high porosity and permeability of

2

the scaffold. These parameters as well as interconnectivity of the pores facilitate the migration of the cells

3

into the scaffold and allow the exchange of cytokines between PHHs during the culture. A selection of

4

different scaffold types that were successfully used for PHH culture is summarized in Table 3.

5

Table 3 Types of scaffolds used in PHH culture Type of scaffold

Approach

Result

Reference

Matrigel/ silk scaffold

Improvement of hepatic morphology and functionality Commercial collagenbased scaffold for PHH cultivation and shipment Fabrication by electrospinning. For the long term PHH cultivation

Improved urea production and albumin synthesis over a time period of 10 days compared to 2D culture, increased expression of genes associated with hepatic functions as well as an increased CYP activity. Co-culture with hepatic stellate cells further increase the metabolic function of the cells PHH shipment on the scaffold reduces the PHH loss during transport. PHH cultivation on the scaffold improves the function of several CYP enzymes, the albumin synthesis and urea detoxification over 10 days. Disadvantage: detaching of the PHHs for re-seeding or RNA isolation is not possible The PLGA scaffold cross-linked with collagen is superior in terms of increasing PHH metabolic activity to the PHH sandwich culture. The production of albumin and urea as well as the expression of CYP enzymes (CYP 3A4 and CYP 2C9) are significantly increased over 14 days compared to sandwich culture. Disadvantage: no increase in the activity of the CYP enzymes compared to the sandwich culture. The PHHs are taken up in an alginate solution and printed into a 3D architecture by 3D printer. Compared to 2D, a higher urea and albumin production was seen after 7 days of cultivation. The additional integration of MSCs into the scaffold further increased the PHHs metabolic activity and the expression of hepatic genes. Disadvantage: this approach is very complex

[118]

Cryogel

Nano-fibrous PLGA (poly(L-lactide-coglycolide)) scaffold

3D bio-printed alginate scaffold

3D bio-printing

[34]

[119]

[120]

6 7

The use of decellularized liver tissue is also suitable for the cultivation of PHH. The physical properties

8

such as pore size, stiffness, etc., as well as ECM proteins, are largely consistent with the in vivo situation

9

[121]. As several studies showed, decellularization and recolonization with cells are possible [121,122],

10

but the matrix of the decellularized liver has a small pore size which acts as a significant limitation

11

hampering re-colonization and sufficient nutrients supply. Since it is difficult to modify the physical

12

characteristics of the human liver, alternatives like synthetic scaffolds are often utilized to allow for

13

modifications. One approach is the functionalization of scaffolds by using ECM proteins derived from a

14

decellularized liver. In one study, a poly (NiPAAm)-chitosan-based cryogel was coated with a solution

15

containing decellularized liver matrix. This coating not only improved the function of tested HepG2 cells,

16

but also had a positive effect on albumin production and urea synthesis of PHHs [123]. Moreover, the 22

1

purified and digested decellularized extracellular liver matrix can be mixed with polycaprolactone (PCL).

2

This mixture can be used as a bioink for scaffold patterning by using a 3D printer. HepG2 cells plated on

3

the generated scaffolds showed an enhanced expression of hepatic marker genes, such as HNF4α, as

4

well as an improved production of albumin and urea compared to cells on collagen scaffolds [124]. In the

5

same lime of evidence, it was shown that, ECM from the decellularized liver could be used for

6

electrospinning of a gelatin/ PCL-based nanofiber to produce ECM nanofiber scaffolds. The culture of

7

PHHs onto such scaffolds increased their function [125].

8

As described before, the culture of PHH on scaffolds has some limitations. There are various scaffolds

9

composed of different components, including some based on natural products like collagen or liver ECM,

10

which makes standardization very challenging. Another restraint is the extraction of cells or their RNA as

11

well as proteins from the scaffolds, which renders the analysis of cells very difficult. Additional challenges

12

of scaffold cultures are the microscopically monitoring of the cells and adaptation for high-throughput

13

screening [126].

14

3.3 Scaffold-free 3D micro tissues

15

3.3.1 Spheroid cultures of PHHs

16

Spheroid cultures can be defined as a cell culture technique, in which an aggregate of cells is formed

17

independently from an exogenous matrix [127]. The first report of liver cell aggregates has been described

18

in 1985, clearly demonstrated that liver cells plated on non-adherent plastic re-aggregated spontaneously

19

and form spheroidal aggregates with a diameter of up to 150-175 µm [128]. The cell culture in spheroids

20

has been further evolved over the past 30 years. In particular, new methods for generating the spheroids,

21

such as the hanging drop technique, the encapsulation of cells in a matrix, the levitation of the spheroids

22

by using a magnetic field, etc., were developed [129]. An advantage of spheroid cultures are the

23

requirement of only 1330-2000 PHHs per spheroid which is comparable to the amount of cells necessary

24

for a 384- well plate and significantly less compared to other 3D cultivation techniques [130,131]. A recent

25

study claims that PHH cultivation throughout 5 weeks is possible and the CYP activity remains almost

26

unchanged between day 8 and day 35. Unfortunately, no comparison to the initial CYP activity was

27

reported in this publication, which makes the interpretation of these results difficult. The authors claim that 23

1

the proteome of the 3D spheroids closely resembles the in vivo liver at the proteome level even after 7

2

days, however, their data clearly show that the expression of more than 130 proteins are de-regulated

3

compared to the in vivo situation [132]. In another recent study, PHHs in spheroid culture were compared

4

to the freshly thawed PHHs as well as to plated PHHs at day 0. A proteome analysis showed that the

5

enzymes responsible for the absorption, distribution, metabolism and excretion of drugs are better

6

preserved in spheroid cultures over 14 days compared to 3D sandwich cultures [133]. However, the study

7

also raises questions as for example the fact that day 0 defines the time at which the metabolic tests were

8

started, not the day the cells were plated. Accordingly, the first measurement day in the sandwich culture

9

takes place 48 hours after plating/isolation. At this time point the PHHs are usually already at a relatively

10

low metabolic level [90]. In addition, there is no comparison of the viability between the two cultivation

11

techniques, therefore, no real comparison of the two cultivation techniques is really possible. Overall, the

12

generation of liver spheroids is often referred to as self-organizing liver tissues, suggesting that it can

13

mimic the organ morphology [134]. However, the liver is much more complex than a cell clump, and no

14

necrotic areas are known in the healthy liver. In addition, hepatocytes with their basolateral side have

15

contact to the blood and on the apical side the bile flows off, which is a main characteristic of the complex

16

liver lobule architecture [135] that cannot be reproduced in a spheroid, yet.

17

3.3.2 Organoid culture of liver cells

18

In contrast to spheroid cultures which mainly aim to maintain the function of already differentiated cells

19

within the mini-tissue, progenitor cells proliferate and self-organize in the ‘organoid culture organ’ and

20

differentiate to some degree into an organ-like tissue [136]. Further differences between spheroid and

21

organoid culture are summarized in Table 4.

22

The organoid formation occurs by mimicking molecular pathways, that are known from embryogenesis

23

and tissue repair in vivo [136,137]. Regeneration of liver tissue can be described by two different models.

24

The first model refers to chronic liver injury, where all hepatocytes are affected. In this model proliferation

25

and differentiation of small Leucine-rich repeat-containing G-protein coupled receptor 5 positive progenitor

26

cells (Lgr5+) are observed near the bile ducts. The second model describes massive proliferation of

27

mature hepatocytes in vivo, which happens after an acute liver injury such as a partial hepatectomy. Both

28

processes have been reconstructed in vitro and resulted, after a proliferation and differentiation phase, in 24

1

liver organoids [138,139]. It has been described that organoid formation triggered by Lgr5+ progenitor

2

cells and long-term cultivation of liver organoids, maintain some liver specific markers, like CYP 3A4

3

activity, ammonia elimination as well as albumin secretion. Although an ‘increase in the differentiation’ of

4

the organoids was reported, it must be critically noted that the authors compared their organoid data to

5

HepG2 or HepaRG cell lines and not with freshly isolated PHHs [140]. Nevertheless, the authors could

6

show albumin and HNF4α expression, as well as glycogen storage in organoids. The albumin secretion

7

and CYP 3A4 enzyme activity was in the range of PHHs, although a direct comparison is gain difficult

8

since the culture condition for PHHs are not clearly described [138].

9

In summary, the organoid technology is an interesting and promising technology for the generation and

10

maintenance of differentiated and functional PHHs [141]. However, this technology has two main

11

disadvantages: the organoid culture is very time consuming and expensive [142]. Nevertheless, organoids

12

may play an important role as in vitro liver toxicity models in the future.

13

Table 4 Comparison of spheroid and organoid culture approaches

Used cell type Aim Used cultivation technique

Spheroid

Organoid

Mature cells Maintaining the differentiation of mature cells by using their natural tendency to aggregate Usage of techniques which not allow the adherence of the cells

Stem cells/ precursor cells Recapitulating embryonic development or cellular processes that take place during tissue repair Cultivation on Matrigel

Used additives

Normal culture media without special additives

Degree of differentiation

The cells remain in a differentiated status over a longer period

Usage of cytokines and growth factors which are necessary for the differentiation Initially low, a certain degree of differentiation can be achieved during the differentiation process

Cultivation time

≤ 5 weeks

≤ 11 months

References

[15,132,143,144]

[136,138-140,145]

14

25

1

4. Advanced approaches for the cultivation of PHH

2

4.1 Miniaturization and combination of co-cultures

3

In the previous sections, many interesting approaches to improve the cultivation of PHHs have been

4

presented. Over the past decades, parallel developments, like combinations of different approaches such

5

as 3D co-culture as well as the miniaturization of various culture models took place [9,109,146]. The

6

newest trends seem to be perfusion-incubator-liver-chips including temperature control, the pH and the

7

media-flow [147]. In this set-up rat hepatocyte spheroids have been cultured up to 14 days without an

8

additional incubator. The results showed a higher albumin and urea production as well as a higher

9

enzyme activity in the fluid-flow system compared to ‘simple’ monolayer or sandwich cultures but also

10

compared to static spheroid cultures [147]. Moreover, the co-cultures of different liver cells on scaffolds or

11

in a 3D spheroid culture was successfully tested leading to increased gene expression of albumin and

12

CYP 3A4 as well as in albumin production compared to 2D or monocultures [130].

13

4.2 The liver is not an isolated organ - various organ-on-a-chip technology approaches

14

We described above the newly developed co-culture models in combination with new scaffolds, or models

15

cultured in fluid-flow technologies. However, in order to study the effects of liver metabolites on different

16

organs, new interesting challenges remain to be solved. How such a cultivation system may look like was

17

shown by Ishida 2018 and Ronaldson-Bouchard et al. 2018 [78,79] and is summarized in Figure 6. Due to

18

the very high complexity of the different organ systems and the different cell requirements concerning the

19

culture conditions, many open questions remain yet unsolved.

20

26

1 2

Figure 6 Possible multi-organ fluid-flow in vitro model, adapted from [148,149]

3

In one of these models, the interaction between PHHs and cardiomyocytes was investigated by placing

4

both cell types on a multi-organ chip. One of the advantages of this model is serum-free cultivation.

5

Cyclophosphamide and terfenadine were tested as model substances for cardiotoxicity. In vivo, the parent

6

drug cyclophosphamide is non-cardiotoxic, but it is converted into a cardiotoxic metabolite in the liver.

7

Terfenadine acts in an opposite manner, the parent compound is cardiotoxic but the metabolite is not. The

8

proposed model was able to demonstrate that this multi-organ chip can initiate toxicity as well as

9

detoxification of different substances [150]. Of course the limitation of each individual cell system will

10

remain, when several of them are combined in a flow system. A further limitation is the relatively high

11

effort required for multi-organ fluid-flow in vitro models, which hampers the analysis of larger numbers of

12

compounds.

13

In another approach, it was tested whether the formation of a reactive metabolite and the activation of four

14

skin sensitization responsible immune cells can be tested in an organ-on-a-chip approach. The liver

15

compartment and the immune compartment are connected in this approach by thin nanotubes. Three

16

drugs known for skin sensitization, including carbamazepine were tested. The liver cells (HepaRG) were

17

able to form the corresponding reactive metabolites of the substances. In particular, in the organ-on-a-chip 27

1

approach, these metabolites could activate the immune cells (U937), which was demonstrated by

2

increased expression of interleukin (IL) 8, IL-1β and CD86. A limitation of the study is that it also comes to

3

an exchange of the different kinds of medium between the compartments through the thin nanotubes,

4

which has an influence on the activation of immune cells, but this cannot be prevented, since such an

5

exchange is necessary if the medium active metabolite is needed [151].

6

4.3 Animal models of PHH engraftment

7

Another interesting approach for the development of a predictive preclinical test system for testing new

8

drugs is the establishment of ‘humanized’ mouse models. Immunodeficient mice with specific knockouts

9

such as fumarylacetoacetate hydrolase (FAH) are frequently used used for this purposes. Briefly, a FAH

10

gene defect leads to the accumulation of toxic tyrosine metabolites in the mouse liver and subsequently to

11

the destruction of host hepatocytes in mice. During this process injection of human hepatocytes with an

12

intact FAH gene can migrate to the liver and colonize the mouse liver. Using this technique an in vivo

13

model can be generated that theoretically represents the complete metabolic properties of the human

14

liver. The extent to which such a model can predict the hepatotoxicity of new drugs will still have to be

15

investigated in further studies [152-154].

16

5. Limitations of Hepatocyte Cultivation

17

As it has been shown in various studies for almost 40 years, cultivation of hepatocytes over some period

18

and maintenance of some functions is possible [155,156]. Nevertheless, as it can be depicted from Figure

19

7, more than 14,000 publications dealing with the cultivation of hepatocytes since 1980, indicating that

20

intensive research is still being carried out in this area.

21

28

1 2

Figure 7 Publications found in PubMed using the term hepatocyte culture

3

As already pointed out many of the approaches in the past decades aimed to maintain the metabolic

4

activity of cells for extended periods. As lined out before many recent approaches aim at mimicking the in

5

vivo environment as close as possible to provide the cells with an adequate microenvironment. The most

6

important techniques for cultivation of PHH that are discussed in this review, as well as their functionality

7

and possible duration of cultivation, are summarized in Figure 8.

8

29

Liver slices Suspension culture

Functionality

Organ-on-a- chip Spheroid Culture Co-Culture 3D Scaffold Culture 3D Fluid-Flow Culture

2D culture

Organoid Culture

Cultivation period 1 2

Figure 8 Functionality and possible duration of cultivation of different techniques PHH cultivation approaches

3 4

Although so many partly highly complex models for the cultivation of hepatocytes are available, they have

5

all in common that the outcome of maintaining all metabolic activities over longer time periods is still

6

unsolved. However, the main question is: why do some studies claim that the metabolic activity can be

7

maintained for several weeks or even months? These apparent contradictions might be explained by the

8

corresponding reference culture used. As already shown by different studies, there is a massive de-

9

regulation of the membrane proteins, during the isolation of cells and in the hours thereafter [157-159]. A

10

proteome analysis has clearly shown that 457 proteins were significantly de-regulated after 24 hours in

11

culture compared to the ‘intact’ liver tissue. Interestingly, the number of de-regulated proteins in 2D culture

12

after 7 days decreased to 358 proteins and in spheroid culture to 132 proteins [132]. This suggests that

13

the isolation of the cells per se seems to be a major challenge although a limited recovery may be

14

possible afterwards [160]. The use of controls where changes have already been induced, e.g.; by the

15

isolation and/or a short period of cultivation, may explain why no major differences can be detected. A

16

significant improvement in the metabolic activity, as reported in several studies, should therefore be

17

critically be discussed. Ideally, PHHs and liver tissue directly shock-frozen after surgery from several 30

1

donors should be used for comparison. It would also be useful to define generally binding reference

2

values obtained by standard methods.

3

Conclusion

4

Drug-induced liver injury is a major etiology of acute liver failure. More than 1000 commercialized drugs

5

can cause severe liver injury. This demonstrates the importance of generating a reproducible and easy

6

accessible platform for drug screening that reflects the liver. The use of human hepatocytes still remains

7

the gold standard for in vitro testing of new drugs. Despite significant approaches, there is currently no

8

technique available, which could be used as a standard model with the ability to predict the hepatotoxicity

9

of new drugs over a longer period of time. However, with the introduction of spheroid or organoid-like

10

culture approaches, or even more complex technics, such as the organ-on-a-chip cultures or bioprinted

11

livers, are some promising directions in research, that shall be further explored to establish a predictive in

12

vitro hepatocyte models in the future. Nevertheless, due to the high complexity of such systems it is

13

strongly adviced to use more interdisciplinary cooperations to achieve a reliable system that reflects liver.

14

In addition, a standardization of

15

necessary to determine the quality of each system.

16

Acknowledgments

17

We would like to thank Svetlana Gasimova and Andrew McCaffrey for editing the manuscript. We also

18

would like to thank Dr. Jürgen Kraut and Silas Rebholz from the Hochschule Esslingen, University of

19

Applied Sciences, Esslingen, Germany who performed the SEM images of the liver tissue. This study was

20

partially funded by the Federal Ministry for Economic Affairs and Energy within the framework of the ZIM

21

program (ZF4301401CS6) and the Ministry for Rural Areas and Consumer Protection Baden-Württemberg

22

(BW 05110214)

23

Conflicts of Interest

24

The authors declare no conflict of interest.

31

test batteries for testing the quality of new in vitro models will be

1

Abbreviations 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

HEPES

5-azacytidine

5-AZA

Atomic force microscopy

AFM

Aurora kinase A

AURKA

Cysteine-rich protein 2-binding protein

CSRP2BP

Cytochrom P450

CYP

Epithelial-Mesenchymal Transition

EMT

Establishment of sister chromatid cohesion NAcetyltransferase 2

ESCO2

Extracellular Matrix

ECM

Extracellular Signal-Regulated Kinase

ERK

Fetal calf serum

FCS

Fumarylacetoacetate hydrolase

FAH

Food and Drug Administration

FDA

Hepatocyte nuclear factor 4 alpha

HNF4α

human papilloma virus

HPV

human telomerase reverse transcriptase

hTERT

Interleukin

IL

Lipopolysaccharides

LPS

Liver sinusoidal endothelial cells

LSECs

Mitogen-activated protein kinase

MEK

Non-Parenchymal Cells

NPCs

Oncostatin M

OSM

32

Polycaprolactone

PCL

Primary Human Hepatocytes

PHHs

Scanning Electron Microscope

SEM

SET Domain Bifurcated Histone Lysine Methyltransferase 2

SETDB2

small Leucine-rich repeat-containing G-protein coupled receptor 5 positive progenitor cells

Lgr5+

Williams E

WE

1 2

33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

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Highlights • • • •

Drug-induced liver injury is a major etiology of acute liver failure Human hepatocytes are the gold standard for in vitro testing of new drugs Currently, there are no techniques available to predict the hepatotoxicity of new drugs over a longer time period Some promising approaches for the prediction of hepatotoxicity exists, but these need to be further developed to establish a predictive in vitro model in the future.

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: