Hypothermic storage of human hepatocytes for transplantation

Copyright © 2013 Cognizant Communication Corporation

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CT‐1055 Cell Transplantation Epub; provisional acceptance 05/10/2013

DOI: 10.3727/096368913X668627

CT-1055 Accepted 05/22/2013 for publication in “Cell Transplantation”

Hypothermic storage of human hepatocytes for transplantation.

Roberto Gramignoli1, Kenneth Dorko2, Veysel Tahan2, Kristen J. Skvorak2, Ewa Ellis3, Carl

Jorns3, Bo-Goran Ericzon3, Ira J. Fox4, Stephen C. Strom1,*.

1 Department of Laboratory Medicine, Division of Pathology, Karolinska University Hospital,

Stockholm, Sweden

2 Department of Pathology, School of Medicine, University of Pittsburgh, Pittsburgh, PA, USA

3 Department of Clinical Science, Intervention and Technology (CLINTEC), Division of

Transplantation Surgery, Karolinska University Hospital, Stockholm, Sweden

4 Department of Surgery, Children’s Hospital of Pittsburgh, University of Pittsburgh,

Pittsburgh PA, USA

*Corresponding author: Stephen C. Strom, Ph.D., Department of Laboratory Medicine,

Division of Pathology, Karolinska University Hospital, SE-141 86 Huddinge,

Stockholm, Sweden

[email protected]

Running title: Hypothermic storage of human hepatocytes

Key words: Hepatocyte transplantation, human hepatocytes preservation, cold storage,

hepatic functions

mailto:[email protected]


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Abbreviations:

BNF - beta-Naphthoflavone

CYP – Cytochrome P450

EROD - Ethoxyresorufin-O-Deethylase

HMM – Hepatocyte Maintenance Medium

HTx – Hepatocyte Transplant

LCU - Luminescent Counting Unit

OLT – Orthotopic Liver Transplantation

PB - Phenobarbital

PE - Plating Efficiency

Rif – Rifampicin

UW – University of Wisconsin solution


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ABSTRACT

Transplantation of human hepatocytes is gaining recognition as a bridge or an alternative to

orthotopic liver transplantation for patients with acute liver failure and genetic defects. Since

most patients require multiple cell infusions over an extended period of time, we investigated

hepatic functions in cells maintained in University of Wisconsin solution at 4°C up to 72h.

Eleven different assessments of hepatic viability and function were investigated both pre and

post hypothermic storage, including plating efficiency, caspase 3/7 activity, ammonia

metabolism and drug metabolizing capacity of isolated hepatocytes. Long-term function,

basal and induced cytochrome P450 activities were measured after exposure to prototypical

inducing agents. Cells from 47 different human liver specimens were analyzed. Viability

significantly decreased in cells cold-stored in UW solution, while apoptosis level and plating

efficiency were not significantly different from fresh cells. Luminescent and fluorescent

methods assessed phase I and II activities both pre and post 24-72h of cold preservation. A

robust induction (up to 200-fold) of phase I enzymes was observed in cultured cells. Phase II

and ammonia metabolism remained stable during hypothermic storage although the

inductive effect of culture on each metabolic activity was eventually lost. Using techniques

that characterize 11 measurements of hepatic viability and function from plating efficiency, to

ammonia metabolism, to phase I and II drug metabolism, it was determined that while

viability decreased, the remaining viable cells in cold-stored suspensions retained critical

hepatic functions for up to 48 h at levels not significantly different from those observed in

freshly isolated cells.


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INTRODUCTION:

Hepatocyte transplantation (HTx) is gaining acceptance as an alternative therapy, in

particular for patients with inherited metabolic liver diseases. These diseases are

characterized by lack of one particular enzyme or protein, giving rise to hepatic and/or

extrahepatic disease. As the liver has a high redundancy in function, replacement of 5-20%

of the liver with healthy donor hepatocytes could correct a wide range of inherited metabolic

liver diseases (8). However, due to the risk of portal thrombosis, only 5% of the liver mass

can be infused at one transplant session (9). In current clinical protocols, hepatocytes are

transplanted by intraportal infusion over a period that may last up to 48 hours (9,15,19). As a

result, hepatocytes need to be cold stored between repeated infusions (9). The deterioration

of the integrity and function of hepatocytes in suspension after a few hours of incubation at

37°C is well recognized, while the beneficial effect of the hypothermic preservation of

hepatocytes in culture media (i.e. Leibowitz or Williams E solutions)(7,20,26,31), or

University of Wisconsin (UW), histidine-tryptophan-ketoglutarate, and Celsior solutions, has

only been partially investigated (11,14,18,23,31).

Although hypothermia has a protective effect due to lowering metabolic functions, cold

ischemic damage is evident upon rewarming (3,14). Belzer’s or UW solution was originally

developed for organ/tissue preservation and is currently the preferred solution for cold

storage of isolated hepatocytes (4), and has been used for long distance shipments of

hepatocytes between the isolation center and transplantation sites (9). This solution has

been adapted to the intracellular ion composition, with a high buffering capacity and contains

impermeable agents to help maintain osmotic gradients through cold storage. (4). Because

of these properties, UW is the solution included for cold storage of hepatocytes in the clinical

application submitted from our institutions to the regulatory authorities. It is known that the

time span for preservation for whole organs is limited to several hours. The time limit for cold

preservation of isolated hepatocytes is not known.


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There are a number of investigations of the effect of cold ischemia-warm reperfusion injury

and the underlying mechanisms involved in hepatocyte cell death (16,22,28) but mainly for

experimental purposes. How the duration of cold storage of hepatocytes affects the quality of

cells for clinical cell infusion has not been systematically investigated.

In order to satisfy regulatory requirement for clinical transplants, one has to demonstrate that

a specific step in the process such as cold storage of cells prior to transplantation still results

in cells with adequate function to be useful for transplantation. In this study we provide data

on hepatic function from short-term and longer-term experiments from cells cold-stored in

UW solution for up to 72 h., from up to 47 human hepatocyte isolations. The viability and

function of cold stored hepatocytes were analyzed using the same procedures employed at

our center to assess the suitability of hepatocytes for clinical transplants. The results

indicate that critical hepatic functions are well maintained in hepatocytes cold-stored in UW

solution for up to 48 h. The data obtained from these studies satisfy the regulatory

requirement to demonstrate function in the cold-stored cells and provides a scientific basis to

support the use of hepatocytes for clinical transplants for up to 48 h post isolation.

MATERIALS AND METHODS

Hepatocyte Isolation.

All tissues were collected with informed consent (IRB protocol 0411142, University of

Pittsburgh, PA, USA). Hepatocytes were isolated from 47 different donors (Table 1): 19

samples obtained from organ donors rejected for Orthotopic Liver Transplantation (OLT) for

reasons including prolonged warm/cold ischemia time, drug overdose, anoxia, steatosis and

fibrosis; 28 specimens derived from patients undergoing scheduled liver resection

procedures performed for a number of different forms of neoplastic disease or undergoing

OLT for genetic inborn errors. Whole donor organs not used for OLT were flushed, in situ


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with cold preservation solution and maintained on wet ice and received for cell isolation 6-36

hours post-cross-clamp. Residual liver tissue not needed for diagnostic purposes was

transported to the laboratory from the operating rooms in ice cold UW or Eagle’s Minimum

Essential Medium (Lonza, Walkersville, MD), respectively and perfused within 90 minutes of

removal.

The tissue dissociation and subsequent hepatocyte isolation procedures were

performed as previously described (12). Cell viability, assessed by trypan blue exclusion

method, and plating efficiency were performed as previously described (12).

For the purpose of this study, cells were analyzed immediately after isolation and

after 24, 48 and 72h in UW solution (concentration of 2-4 x 106 viable cells/ml) at 4oC. Every

24h an aliquot of cells was diluted 1:3 with cold Eagle’s Minimum Essential Medium and

centrifuged at 80 x g for 5 min and resuspended in plating medium, as previously described

(12).

Caspase-Glo™ assays.

Caspase-3 and Caspase-7 were measured with a luminescent assay (Caspase-Glo®

3/7, Promega Corporation, Madison, WI, U.S.A.) according to the manufacturer’s instructions

with some minor changes as described here. Briefly, 40 l of the cell suspension were

seeded in a 96-well white plate and let equilibrated for 30min at 37oC. An equal volume of

Caspase-Glo™ reagent was then added directly to the well. After 30min incubation at room

temperature, luminescence was read directly on a white plate in a luminometer (Synergy HT,

BioTek Instruments, Winooski, VT) with an integration time of 1 second/well. Luminescence

produced by the assay is proportional to the amount of caspase activity present in the cell

sample. Results are expressed as LCU/min and normalized to one million viable cells.

Drug Metabolism Studies.


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Cytochrome P450 (CYP) activities were measured immediately after washing while

the cells were in suspension and after plating and attachment. Methods to assess ammonia,

testosterone and 7-ethoxyresorufin metabolism (CYP3A4 and 1A1/2), resorufin conjugation,

media and culture conditions were as previously described (12). Additional cell-based

assays for specific CYPs (2C9; 3A7; 3A4) were used according to manufacturer´s

instructions with minor modification. Briefly, 3 x 104 hepatocytes were added to a 96-well

white plate and incubated for 30 minutes at 37°C with pro-luminogenic compounds for

specific CYPs (CYP-Glo™, Promega Corporation). The luminogenic product was detected

with a Luciferin detection reagent and after incubation for 20 min at room temperature

luminescence was read directly in a white plate in a luminometer (Synergy HT) with an

integration time of 1 second/well. Attached cells were analyzed after 5 days in culture by

exposing the cells in 96-well clear culture plate to different Glo™ substrates and transferring

the suspension after lysing method to a 96 white plate and read with the same protocol. The

luminescence produced is proportional to CYP activity. Results are expressed as

Luminescent Counting Units (LCU)/min and normalized to one million viable cells or to the

dsDNA content.

dsDNA amount quantification.

Double-stranded DNA (dsDNA) quantification was performed with a Quant-iT™

PicoGreen® dsDNA ultrasensitive fluorescent nucleic acid staining kit according

manufacturer’s instructions (Molecular Probes, Invitrogen Corporation, Camarillo, CA). After

the Glo™-assays were complete, each well was incubated with 100 l Quant-iT™

PicoGreen® in TE buffer and the fluorescence intensity was read on a fluorescent

spectrometer (Synergy HT) at an excitation wavelength of 488/15 nm and an emission

wavelength of 528/20 nm. dsDNA concentration was quantified by interpolating the A528

values for the unknowns from a standard curve of lambda DNA using the equation (dsDNA

[mg/ml] = 0.1057 * A528 – 61.322; R2 = 0.9941).


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Statistical analysis.

Experiments were performed in triplicates. Results are expressed as mean ±

standard error of the mean (SEM). Unpaired t-test on pair group and analysis of variance

(ANOVA) with Newman-Keuls multiple comparison test has been used to determine

statistical differences. p value < 0.05 was chosen as the minimum level of significance.

Results are presented as histograms showing mean ± SEM. All data have been analyzed by

GraphPad Prism software (version 5.03, GraphPad Software Inc.)

RESULTS:

During the study period, 47 liver tissues were collected and successful hepatocyte isolations

were performed on all specimens. The study group was composed by patients undergoing

liver resection due to primary or secondary malignancy (n=18), liver transplantation due to

metabolic liver diseases (n=10), or organ donors rejected from organ transplant for different

reasons (n=19) (Table 1). The donors were equally distributed between females (47%) and

males (53%), age ranged from 0.6 to 85 years (39 ± 4 years).

After isolation, hepatocytes were analyzed directly (Fresh) or analyzed after 24, 48, 72 hours

of cold storage in UW solution. Freshly isolated hepatocytes showed a mean viability of 81 ±

1%. A significant decrease in term of viability was measured every 24 hours of cold storage:

61 ± 2% at 24h, 52 ± 3% at 48h and 48 ± 3% at 72h (p < 0.0001) (Fig. 1A). The cell recovery

after cold storage ranged from 50-75% of the total viable cell number initially added to

aliquots.

The level of apoptosis, quantified by Caspase 3 and 7 activities, was evaluated in 23 of the

preparations. A trend towards increased caspase activity was observed with longer cold

storage, but differences did not reach statistical significance (Fig. 1B).


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The ability of hepatocytes to attach to a cell culture plate coated with extracellular matrix

requires that viable cells express functional cell adhesion molecules and maintain sufficient

ATP levels to maintain adherence (29). All hepatocyte suspensions showed high plating

efficiencies on collagen substrate post cold storage, and again, there was no statistical

difference in PE between the cold-stored and freshly isolated cells (Fig. 1C). Once attached

to the substrate, all cellular preparations showed typical primary human hepatocyte

morphological (polygonal shape, one or more nuclei, granular cytoplasm with small vesicular

inclusions and bright sharp cell membranes surrounded and canalicular structures) without

any noticeable differences between pre- and post-hypothermic storage (Fig. 1D). After two

days of culture, all the cell preparations formed a confluent or semi-confluent monolayer on

collagen coated plates.

Cytochrome P450 activities

Since transplanted cells would be expected to perform metabolic functions immediately post

transplantation, activities mediated by CYP 1A1/2, 2C9, 3A4 and 3A7 were analyzed.

CYP1A1/2 showed stable activity during the first 48h of cold preservation and a slight, but

not significant, increase in activity after 72 h of cold preservation (Fig. 2A)(p=0.42). Since the

toxic effects of long-term cold storage might not be obvious from analysis conducted

immediately after recovery from cold storage, we also investigated long-term hepatocyte

viability and function by plating the cells on collagen and analyzing basal and induced levels

of CYP activities. Basal EROD activity and the induction in response to exposure to the

prototypical inducer (BNF) was investigated (Fig. 2B). Exposure to BNF for 3 days induced

up to a 24-fold increase in EROD activity over control (p<0.0001).

Activities mediated by CYP3A were maintained through cold preservation. When CYP3A4

mediated metabolism was assessed immediately after isolation, an activity equal to 16,739 ±


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Luciferin H metabolism is specifically mediated by 2C9 in human hepatocytes. Although a

trend towards decreasing 2C9 activity was noted, the results in cold stored samples were not

significantly different from fresh cells (Fig. 2F).

2,644 LCU/min/million of hepatocytes was present (Fig. 2C). After 24h in UW a slight but not

significant increase in activity in the cells was measured (23,894 ± 8,045 LCU/min/million;

p=0.54). Activity remained stable and not statistically different from freshly isolated cells in

hepatocytes recovered after up to 48h, or 72h of cold storage (Fig.2D). A 3-day prior

exposure to rifampicin induced significant increases in CYP3A activities in fresh, 24h and

48h cold-stored cells. Cells stored for 72h. showed an increase in activity, although the data

did not achieve statistical significance. The results were the same as those with the

luminescent assay when 3A4 activity was measured with the traditional HPLC method to

detect the 6-beta-hydroxy metabolite of testosterone (data not shown).

Activities mediated by CYP3A7 showed a profile similar to that observed with CYP3A4

(Fig.2E). As expected, in cells obtained from patients younger than 3 years in age (n=7),

activity of the fetal 3A, 3A7, was prominent.

Phase II activity

Conjugation of resorufin is a measure of phase II metabolic activity, and was evaluated in

immediately after recovery in cold-stored cells and also after attachment with 5 days in

culture. There was no statistical difference in resorufin conjugation between fresh and cold

preserved cells (Fig. 3A). Conjugation activities are generally higher in plated cells (hatched

bars), than those measured in suspension (open bars). This inductive effect on conjugation

activity was observed in cells from nearly all human cases. A significant increase in

conjugation activity in cultured cells was observed maintained out to 48h of cold storage (Fig.

3A). At 72h of cold storage there was a slight, but non-significant increase in conjugation

activity in plated cells.


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Ammonia metabolism

Like conjugation capacity, ammonia metabolism is generally greater in fresh cells or

hepatocytes maintained in culture for several days as compared to those in suspension.

There was no difference in ammonia metabolism between fresh and cold stored hepatocytes

when measurements were made immediately after recovery (Fig 3B, open bars). A

progressive and significant decline in ammonia removal was observed in cells kept at 4°C

past 24h. when they were plated and analyzed 5 days post plating (Fig. 3B, hatched bars).

DISCUSSION:

There are a large number of patients awaiting liver transplantation, since the availability of

donor organs is limited. Hepatocyte transplantation is a promising therapy for a variety of

indications (5,8,21,24). A typical protocol will require infusion of billions of hepatocytes. The

total number of hepatocytes injected in a HTx event is calculated to be in the range of 1-2 x

108 per kg body weight (6,10). Because of the risk of portal thrombosis (2) from a single

infusion of a large number of cells, the target cell number is frequently split into 3-5 separate

infusions over 24-36h. (9,10). As a result isolated hepatocytes need to be stored from

several hours to several days prior to transplantation (9,27). During that time, isolated

hepatocytes are usually cold preserved (2° - 8°C) until needed. Moreover, currently revised

clinical protocols now include pre-treatment of the recipient with local radiation (10) or partial

liver resection (21). Since preconditioning of the recipient is not begun until it is clear that

sufficient numbers of isolated hepatocytes will be available to complete the transplant,

preconditioning can add several more hours of cold-time post isolation. To date, there is no

general consensus on an optimal protocol to maintain cells, post isolation and prior to

transplantation. In this study UW was chosen as the preservation solution since it has been

previously used for storage of human hepatocytes for transport between isolation and


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transplant centers (9) and for its documented superior properties in preserving human

hepatocytes compared to other organ preservation solutions (1). Because of these

properties, UW was the only solution included in the protocol submitted from our institution to

the Food and Drug Administration for the cold storage of hepatocytes prior to transplant (10).

The purpose of the study was to define the time limits of cold storage that still provided

functional hepatocytes for transplantation. Because UW was the only preservation solution

approved in our clinical transplant protocol, and the large numbers of cells required

performing the assays included here, other storage solutions could not be included in the

present study. We chose instead, to provide the data on cold storage in UW on a large

number of hepatocytes isolations (up to 47) to get a full picture of the data one might expect

from future cell isolations. We utilized hepatocytes isolated both from organ donors (at the

moment the only clinical source of human hepatocytes for cell-based therapy) as well as

tissues unsuitable for clinical applications, including tissue from liver resections for neoplasia

and explanted tissues from inborn error patients (Table 1). In a recently published study, we

demonstrated that cells isolated from patients with some metabolic liver diseases provide

highly viable hepatocytes that engraft and repopulate liver when transplanted (13). The

present results with cells isolated from these organs from metabolic disease patients indicate

that these cells weather the stress of hypothermic storage as well or better than cells isolated

from organs rejected for transplantation. In the tissues received from liver resections from

cancer patients, we have not observed any statistical difference in viability, plating efficiency

or drug metabolism between the patients that received chemotherapy, and those that did not.

It is well known that isolated hepatocytes undergo progressive damage and subsequent cell

death during cold storage (1,19,25). Although hypothermia slows cell metabolism and

prevents a rapid loss in viability, extended low temperature storage may cause cellular

damage. Viability, as measured by trypan blue exclusion, was significantly reduced in cold-

stored as compared to fresh cells. A form of apoptosis due to lack of cell anchorage, called

anoikis, may be involved in loss of viable hepatocytes maintained in suspension (25).


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However, in this study of cold-preserved human hepatocytes, only a trend towards increased

caspase 3/7 activities were observed, which did not reach statistical significance even at 72h.

The plating efficiency of the cold-stored cells did not change significantly from that observed

with freshly isolated hepatocytes. These results indicate that although the viability decreased

with extended cold-storage, the ability to attach to a culture substrate is well maintained in

the remaining viable cells through 72h of cold storage.

Biotransformation of drugs or chemical reagents as a measure of hepatic function has been

proposed as a criteria for selection of hepatocyte cases for clinical transplant (9,17). Results

presented here for the CYP 1A, 3A and 2C families support the hypothesis that these

endpoints provide data useful for the analysis of hepatic function prior to transplant.

While most studies focus on CYP450 mediated metabolism, relatively little attention has

been paid to conjugation activity or ammonia metabolism. In addition to phase I metabolism,

long-term phase II and ammonia metabolism are highly relevant endpoints to measure,

particularly since urea cycle disorders and Crigler-Najjar syndrome have been the most

common indications for HTx.

However, for assessing the potentially toxic effects of extended cold storage, the way the

assays are performed becomes quite critical. Except for viability as measured by trypan blue

exclusion, all of the functional assays measured immediately after recovery of the cells from

cold storage show no significant difference between freshly isolated and those stored up to

72h. However, the data presented for CYP3A4 induction and ammonia metabolism in long-

term plated cells suggests that when the previously cold-stored cells are allowed to attach to

culture plates and are maintained for an additional 5 days in culture, subtle, previously latent,

adverse effects of cold storage on hepatic functions are revealed. A significant induction of


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CYP3A4 metabolic activity following exposure of cultured cells to rifampicin is only observed

in cells maintained out to 48 h of cold storage. Likewise, the normal increase in ammonia

metabolism observed in cultured hepatocytes is only statistically significant for freshly

isolated cells and those stored for 24h.

Measurements of CYP1A induction and the conjugation of resorufin were identical to fresh

cells out to 72h of cold storage. The longer-term assays on cultured cells require that the

cells maintain high viability and function over an extended time in culture. Cells not

sufficiently vital to withstand that level of stress are presumed lost to the analysis. Since

these activities were found to decline in long-term cultured cells, measurements of CYP3A4

induction by rifampicin and the increase in ammonia metabolism may be the most sensitive

indicators of cold storage damage to human hepatocytes.

In summary, isolated human hepatocytes were exposed to prolonged storage at 4oC and

examined for viability and function with a battery of 11 tests of that are used in the

investigation of hepatocytes for possible clinical transplant at our centers. This study

supports the hypothesis that isolated hepatocytes can be maintained in UW solution for a

prolonged time without significant loss of most hepatic functions. Long-term culture of

hepatocytes and assays of CYP induction and ammonia metabolism declined with prolonged

cold storage greater than 48h, and may therefore, be more sensitive indicators of cold-

storage damage than assays conducted immediately post cell recovery. While viability

decreased in cold-stored cells to the range of 50-60%, most hepatic functions were well

maintained in the remaining viable cells out to at least 48h post-isolation.


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Acknowledgements: Supported by NIH. N01-DK-7-0004/HHSN26700700004C and

RC1DK086135 (SCS), National PKU Alliance (RG and KJS), COPEV Associazione per la

Prevenzione e Cura dell’Epatite Virale “Beatrice Vitiello” ONLUS (RG).

Disclosures: Authors declare no competing financial interests.


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FIGURE LEGENDS:

Figure 1: Human hepatocyte viability, apoptosis and plating efficiency. Human

hepatocyte viability (A) and caspase activity (B) and plating efficiency (C) for freshly isolated

hepatocytes and cells in UW solution after 24h, 48h and 72h at 4°C. Bars show mean and

standard error. * p<0.01; ** p<0.001; *** p<0.0001. Representative phase contrast

photographs for freshly isolated hepatocytes (D), and cells stored in UW solution for 24h (E),

48h (F) and 72h (G). Photos taken on day 3 of culture, bar represents 50 µm.

Figure 2: Cytochrome P450 activity of human hepatocytes. CYP 1A1/2 measured by the

conversion of 7-ethoxyresorufin to resorufin in cells in suspension immediately after isolation

and after cold storage for 24h, 48h and 72h in UW solution (A), and after plating and 5 days

in culture, including the final 3 days with exposure to (BNF) or vehicle control (DMSO only)

(B). CYP3A4 activity on cells immediately after isolation or recovered from cold storage and

measured in suspension (C) and after culture and a 3 day exposure to Rif measured by

Luciferin-IPA (D). Measurement of CYP3A7 (fetal form) in cells in suspension or immediately

post recovery from cold storage by Luciferin-PFBE (E). CYP2C9 activity measured on cells in

suspension by Luciferin-H (F). Bars show mean and standard error. * p<0.01; *** p<0.0001.

Figure 3: Phase II activity and ammonia metabolism. Resorufin conjugation (A), as a

measurement for phase II activity, and ammonia metabolism (B) were performed with

hepatocytes in suspension (open bars) and with hepatocytes maintained in culture for 5 days

(hatched bars). Bars represent mean and standard error. * p<0.01; ** p<0.001; *** p<0.0001.


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Table 1. Characteristics of the liver tissues investigated. Organ donors were tissues rejected

from OLT for reasons including drug abuse, steatosis or fibrosis, anoxia and prolonged

ischemia time. Residual tissue not needed for diagnostic purposes in case of benign

(adenoma or focal nodular hyperplasia) or malignant diseases (cholangiocarcinoma or

colorectal cancer), and explanted livers from patients undergoing OLT for genetic inborn

errors are also listed. The gender of the donor, the age, the weight of processed tissue and

cell recovery in term of viable hepatocyte per gram of wet tissue, and the viability post-

isolation are reported in the table.

Tissue Gender

(M/F)

Age

(years)

Weight

(gr)

Viability

(%)

Yield

(106/gr)

Organ Donors

Heart Beating Donors 10/3 0.6‐72 325+64 82+6 7.3+7.9

Diseased Cardiac Death 2/2 8‐50 249+119 67+31 5.6+1.8

Prolonged Cold Ischemia Time (24‐36h) 0/2 56‐68 215+91 57+32 1.6+1.0

Liver Resections

Inborn Errors 5/5 0.7‐24 147+66 85+6 8.8+4.0

Benign Diseases 0/3 28‐42 112+16 78+5 3.9+2.3

Malignant Diseases

Primary Tumor 3/2 48‐85 53+21 82+9 17.3+14.6

Secondary Tumor 1/9 22‐79 42+20 78+13 16.0+4.7


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Hypothermic storage of human hepatocytes for transplantation

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