Full Paper

Human Hepatocyte Morphology and Functionsin a Multibore Fiber Bioreactor

Loredana De Bartolo,* Sabrina Morelli, Maria Rende, Carla Campana,Simona Salerno, Nino Quintiero, Enrico Drioli

The viability and liver specific functions of human hepatocytes in a multibore fiber bioreactorare reported. Human hepatocytes were cultured in the intraluminal compartment of thebioreactor. Human hepatocytes on themembranes maintained their round shape and showedfocal adhesions as sites of interaction with themembrane surface. Cells in the bioreactor expressedliver specific functions, including synthetic anddetoxification activity up to 14 d of culture. Theresults demonstrate that human hepatocytes cul-tured in the multibore fiber bioreactor are able tosustain the same in vivo liver functions in vitro.

Introduction

The engineering of human tissue analogues gives a

significant advantage to applied research as differentiated

human tissue ismade accessible for e.g. drug testing, study

of disease and pathology and therapeutics molecules. On

L. De Bartolo, S. Morelli, M. Rende, C. Campana, S. Salerno,N. Quintiero, E. DrioliInstitute on Membrane Technology, National Research Council ofItaly, ITM-CNR, c/o University of Calabria, Via P. Bucci, cubo 17/C,Rende (CS), ItalyFax: þ39 09 8440 2103; E-mail: l.debartolo@itm.cnr.itM. Rende, E. DrioliDepartment of Chemical Engineering andMaterials, University ofCalabria, Via P. Bucci, cubo 45, Rende (CS), Italy

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the contrary to cell lines and conventional culture systems

that frequently induce qualitative changes in the cells,

miniaturized human in vitro tissue analogues provide the

chance to improve human predictability. The development

of both biomaterial and culture technique able tomaintain

liver specific functions could help to realize a systemwhich

comprehensively reproduces human liver functions. Iso-

lated hepatocytes are able to continue the full range of

known in vivo liver specific functions for only a short time

when they are maintained under standard in vitro cell

culture conditions.[1]

Various culture methods have been developed to foster

retention of hepatocyte functions including coculture with

nonparenchymal cells,[2] culture in a sandwich collagen

gel,[3] in three-dimensional systems in spheroids[4] or a

variety of dynamic systems such as bioreactors.[5–12]

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672

Differently from static culture methods, the bioreactors

allow the culture of cells under tissue specific mechanical

forces such as pressure, shear stress and interstitial flow.

Furthermore, in this system a constant turnover of tissue

culture medium augments the gas and nutrient exchange,

which together with the complete fluid dynamics control

ensures the long-term maintenance of cell viability and

functions.[13]

One of the most-used bioreactors for mammalian tissue

growth is the hollow fiber membrane bioreactor. This

bioreactor meets the main requirements for cell culture:

wide area for cell adhesion, oxygen and nutrient transfer,

removal of catabolites and protection from shear

stress.[14,15] Hollow fiber membranes may serve as scaff-

olding material in several ways: 1) initiate tissue

formation, 2) guide the microarchitecture of the develop-

ing tissue by providing histo-appropriate mechanical

forces, 3) confer mechanical strength. The multibore

membrane of modified poly(ethersulfone) (PESM) is

completely new, combining the advantage of having

7 compartments represented by 7 capillaries arranged in

one single fiber so the risk of membrane rupture can be

minimized by using this multibore membrane. Moreover

the 7-hole arrangement enables a high stability to the

foamy support structure that is located in-between the

capillaries and shows a permeability that is higher than

those of other membranes with similar pore size. An

additional advantage of the multibore membrane is the

high chemical andmechanical resistance. This type of fiber

could be useful in the culture and co-culture of cells in

Figure 1. Scheme of the multibore fiber bioreactor and the perfusion system.

several intraluminal compart-

ments and therefore in the

reconstruction of tissue envir-

onment in vitro. We used this

membrane in a bioreactor for

the first time for the culture of

human hepatocytes. Since an

efficient transport of meta-

bolites and nutrients is

required for in vitro mainte-

nance of hepatocyte viability

and functions, we evaluated

the hydraulic permeance and

the mass transfer of metabo-

lites through the membranes.

We investigated the morpho-

logical behavior of cells in the

multibore fiber bioreactor by

confocal laser scanningmicro-

scopy (CLSM) and scanning

electron microscopy (SEM).

The liver specific functions of

human hepatocytes in the

membrane bioreactor have

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been explored with time. In order to investigate the

biotransformation functions of human hepatocytes in

the multibore fiber bioreactor Hypericum perforatum

(St. John’s wort) extract and paracetamol were used.

H. perforatum is a herbal remedy widely used for the

treatment of depression.[16] Among its constituents

hyperforin is one of the major constituents responsible

for antidepressant activity and is biotransformed through

the cytochrome P450 enzyme system. Acetaminophen,

paracetamol or N-acetyl-p-aminophenol (APAP), is an

over-the-counter drug commonly used for its analgesic

and antipyretic properties that is metabolized by hepato-

cytes through glucuronidation and sulfonation reac-

tions.[17] Both components were used to assess the

performance of the human hepatocyte multibore fiber

bioreactor.

Experimental Part

Multibore Fiber Bioreactor

The multibore fiber membrane bioreactor consists of 3 multibore

membranes1 of PESM (Inge AG, Greifenberg, Germany) in order to

increase hydrophilicity and strength of membrane. One single

multibore fiber contains 7 compartments in which cells are seeded.

Thefibers are located in parallelwithin a glasshousing andpotted at

each end in order to establish two separate compartments: an

intraluminal compartment within the fiber, and an extraluminal

compartment or shell outside of the fibers. The two compartments

communicate through the pores in the fiber wall. The total inner

surface area of the multibore membranes used is 1.2 cm2.

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Human Hepatocyte Morphology and Functions in a Multibore Fiber Bioreactor

The bioreactor (volume: 18 mL) was connected to the perfusion

system consisting of a glass medium reservoir, tubing, oxy-

genator, a micro-peristaltic pump and a glass medium waste

(Figure 1).

The medium enters from the reservoir before the oxygenator

where it is heated and oxygenated, then the medium flows into

the membrane bioreactor. The oxygenated medium was con-

tinuously fed to the cells adhered on the membrane in the

bioreactor with a flow rate Q of 0.64 mL �min�1 that was set on

the basis of average retention time. The stream Qout leaving the

bioreactor was monitored and recycled to ensure the continuous

mixing of the medium. Media samples were collected daily from

the outlet stream to evaluate the product formation and the

substrate clearance. Fresh medium was perfused in single-pass

and the stream leaving the bioreactor Qout was collected as waste

until approaching the steady state. When the system reached the

steady state, the stream leaving the bioreactor was recycled (Qr) in

order to obtain the accumulation of products. The fluid dynamics

were characterized without the cells by tracer experiments using

William’s medium. The bioreactor was challenged by changing

the tracer concentration stepwise in the feed stream (Cin) and

the outlet concentration (Cout) was continuously monitored by

on-line spectrophotometer (UV Cord Pharmacia, Uppsala,

Sweden). The bioreactor fluid dynamics were characterized in

terms of the cumulative residence time distribution (RTD) to a step

inputs:

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FðtÞ ¼ Cout=Cin (1)

where t is the actual time.[18]

The experimental mean residence time was calculated as a

mean value of the E curve:

F ¼Z t

0E dt (2)

The theoretical mean residence time was calculated as:

t ¼ V=Q (3)

where Q is the perfusion flow rate and V is the volume of the

bioreactor.[18]

Characterization of Morphological and

Physico-Chemical Properties of the Membrane

Dried multibore membranes were cut in cross-sections, mounted

with double-faced conductive adhesive tape, and sputter-coated

with gold. Treated membranes were analyzed by scanning

electron microscope (SEM, ESEM FEG QUANTA 200, FEI Company,

Oregon, USA). Representative images were acquired to obtain

information about cross-sectional structure and thickness, intra-

and extra-lumen morphology and diameters, and the shape and

size of membrane pores.

The hydrophobic/hydrophilic character of the investigated

membranes was estimated by contact angle technique. Water

contact angles were measured using the sessile drop method

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at ambient temperature by CAM 200 contact angle meter

(KSV Instruments LTD, Helsinki, Finland), depositing the liquid

on the membrane surface using an automatic microsyringe.

Transport Characterization

The mass transport of metabolites through the membranes was

evaluated by a characterization apparatus. The multibore fiber

membranes were potted with an epoxy compound inside glass

modules (length: 20 cm, inner diameter: 1.5 cm) that allowed

access to both the intraluminal and extraluminal compartments.

A peristaltic pump (ISMATEC, General Control, Milan, Italy) circu-

lated themetabolite solution by pumping the fluid (feed) from the

reservoir into the inlet port of the module at flow rates of 0.48–

0.64 mL �min�1. Pressures were monitored at inlet and outlet

of the module by online manometers (Allemano, accuracy:

�0.98 mbar). Inlet pressures were varied in order to obtain

transmembrane pressures (DPTM) from 4 to 70 mbar. The extralu-

minal flow (permeate) was measured continuously, and the

concentration of the metabolite permeating through the mem-

branes was monitored by an online UV-spectrophotometer (LKB

Uvicord SII, Pharmacia, Italy) and/or by assay with phenol in the

case of dextran. Metabolites include albumin (molecular weight

66 kD), immunoglobulin (IgG) (molecular weight 155 kD) and

dextran (molecular weight 473 kD), all purchased from Sigma,

Milan, Italy. Test solutions were prepared by dissolving separately

0.5 mg �mL�1 for albumin, 0.05 mg �mL�1 for IgG and 0.1 mg �mL�1 for dextran in phosphate buffer at pH¼ 7.4.

The multibore fiber module was initially fed with water to

evaluate hydraulic permeance, then it was filled with metabolite

solutions for solute permeation measurements. For each type of

metabolite a new module was used. In order to ensure a high

reproducibility of permeance data, tests were carried out on four

modules and the average hydraulic permeance was reported.

Human Hepatocytes Culture

Primary human hepatocytes (Cambrex Bio Science, Milan, Italy)

isolated from the non-transplantable tissue of young single

donors were used for cell culture experiments. The purity of

isolated hepatocytes is 95% and non-parenchimal cells are present

in a very low percentage (5%). Cryopreserved human hepatocytes

were quickly thawed in a 37 8C water bath with gentle shaking.

Then, the cell suspension was transferred slowly into a tube

containing 25 mL of cold hepatocyte culture medium (HCMTM,

Cambrex Bio Science, Milan, Italy), and centrifuged at 50 g at 4 8Cfor 3 min. The HCMTM is constituted of hepatocyte basal medium

(HBMTM) together with all the components provided in HCMTM

bulletkit1 (Cambrex Bio Science, Milan, Italy): epidermal growth

factors, insulin, ascorbic acid, transferrin, hydrocortisone 21-

hemosuccinate, bovine serum albumin-fat acid free 2% (BSA-FAF)

and gentamicin sulfate 50 mg �mL�1, amphotericin B 50 ng �mL�1.

The cell pellet was suspended in HCMTM and tested for the cell

viability by trypan blue exclusion, as reported elsewhere.[19]

Human hepatocytes were seeded in the lumen of themultibore

fiber bioreactor, previously conditioned with HCMTM containing

BSA, to give a concentration of 1.3� 105 cells � cm�2 and incubated

for the first 24 h at 37 8C in a 5 vol.-% CO2/20 vol.-% O2 atmosphere

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674

with 95% relative humidity in HCMTM containing 2% BSA.

Thereafter, the culture was continued under serum-free condi-

tions for the whole culture time up to 14 d.

The functions of the human hepatocytes cultured in the

bioreactor were investigated in terms of urea synthesis, secretion

of total proteins and biotransformation functions. In particular,

the biotransformation functions were investigated by adding

to culture medium paracetamol (0.3�10�3M) and hyperforin

(1.8 mg �mL�1).

Biochemical Assays

Samples of the culturemediumwere collected from the bioreactor

in pre-chilled tubes and stored at �20 8C until assayed. The urea

concentration was assayed by the enzymatic urease method

(Sentinel, Milan, Italy).

The protein content in the samples was determined by protein

assay using bicinchoninic acid solution (Sigma, Milan, Italy) by

spectrophotometer analysis.

The statistical significance of the experimental results was

established according to the Unpaired Statistical Student’s t-test

and ANOVA test (p< 0.05).

HPLC Analysis of Paracetamol and Metabolites

The samples were HPLC analyzed using LiChrosorb RP-18 column

(5 mm), 250� 4 mm (Merck, Darmstadt, Germany). The mobile

phase consisted of a potassium phosphate buffer (0.5 M) at pH¼6.5 and methanol (95:5 v/v). The flow rate of eluent was

1 mL �min�1 with an analysis time of 30 min at a UV wavelength

of 242 nm. A polymeric column (Strata X, Chemtek Analytica,

Bologna, Italy) was used to treat the samples before injection

into the HPLC system. After conditioning with ethanol and water

Figure 2. Representative scanning electron micrographs of cross-section of multiborePESM membrane.

(1:1, v/v), the samples were acidified with

2% H3PO4 and diluted with potassium

phosphate buffer at pH¼ 6 (1:1, v/v).[20]

The recovery of paracetamol and its meta-

bolites was performed by using two dif-

ferent conditions: the first with 1 mL of

water with 5% of methanol and the second

with 1 mL of methanol. The recovered

fraction was dried under a gentle stream

of nitrogen and it was re-dissolved in 0.2 ml

of water before injecting into the HPLC

system.

HPLC Analysis of Hyperforin

The hyperforin was analyzed using a

LiChrosorb RP-18 column (5 mm), 250�4 mm at a UV wavelength of 270 nm.

Mobile phase A (acetonitrile) and mobile

phase B (2% H3PO4 in water) were used in a

step gradient program at 1 mL �min�1: from

0 min to 10 min a linear change from 15% A

and 85% B to 20% A and 80% B; from 10 min

to 20 min , a linear change from 20% A and

80% B to 75% A and 25% B, and from 20 min

to 25 min a linear change from 75% A and

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25% B to 80% A and 20% B. The samples were diluted with

acetonitrile (1:1, v/v) and after acidification with 0.1 mL of H3PO4,

vortexing for 1 min and then centrifuged at 6 000g for 10 min.[21]

Cell Morphology

Sample Preparation for SEM

Specimens of cell cultures were prepared for SEM by fixation in

2.5% glutaraldehyde, pH¼ 7.4 phosphate buffer, followed by

post-fixation in 1% osmium tetroxide and by progressive

dehydration in ethanol. The specimens were examined by SEM

after plating with gold under vacuum.

Hepatocyte Staining for LCSM

Themorphological behavior of human hepatocytes cultured in the

membrane bioreactor was investigated after 48 h of culture by

LCSM. Hepatocytes cultured in the multibore PESM membranes

were washed with phosphate-buffered saline (PBS), fixed for

15 min in 3% para-formaldehyde in PBS at room temperature,

permeabilized for 5 min with 0.5 % Triton-X100 and saturated for

15 min with 2% normal goat serum (NGS). To visualize vinculin, a

specific monoclonal anti-vinculin antibody (Sigma, Milan, Italy),

diluted 1:50 in 1% NGS, was incubated for 30 min at room

temperature.[22] Then samples were washed twice in PBS and

incubated for 30 min with goat anti-mouse IgG tetramethylrho-

damine isothiocyanate (TRICT) conjugated (Sigma, Milan, Italy),

diluted 1:100 in PBS. To visualize the actin, the samples were

incubated in phalloidin conjugated with fluorescein isothiocya-

nate (FITC) (50 mg �mL�1) (Fluka, Milan, Italy) in PBS. Then samples

were washed twice in PBS and incubated for 20 min with

diamidino-2-phenylindole (DAPI) (Sigma, Milan, Italy) to visualize

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Human Hepatocyte Morphology and Functions in a Multibore Fiber Bioreactor

nucleic acid. Finally, the samples were washed, mounted and

viewed with Laser Confocal Scanning Biological Microscope

(Fluoview FV300, Olympus, Milan, Italy).

Results and Discussion

Properties of the PESM Multibore Membranes

Themorphology ofmultiboremembrane fibers is shown in

Figure 2. One fiber (diameter: 4.1 mm) consists of 7

capillaries with diameters of 960 mm and an inner layer

that is a very thin selective surface. The capillaries are

surrounded by a porous support structure that gives good

stability and mechanical resistance to the fiber. The PESM

membrane has a good surface wettability as the water

contact angle and water sorption measurements demon-

strated (Figure 3). The capillaries have a wettable surface,

in fact the water contact angle measured on this

membrane was 69.5� 2.48 at t¼ 0 s, and the increase of

water sorptionwas about 14% after 10 s. It has been shown

that the surface wettability of the membrane favors

interactions with the cells.[23]

These properties together with hydraulic permeance are

not only important for the attachment of cells but also as

routes for the delivery ofmetabolites towards the cellmass

and the selective removal of secreted products in the

membrane bioreactor.

In this work, experiments aimed at evaluating the

hydraulic permeance of the membranes and the transport

of metabolites like BSA, IgG and dextran, under pressure

gradients, through PESMmembranes were carried out. The

observed steady-state hydraulic permeance of multibore

membranes, calculated as the slope of the flux (J)

versus transmembrane pressure (DPTM) straight line,

Figure 3. Time-related water contact angle (�) and water sorp-tion (&) on multibore PESM membrane surface. The reportedvalues are the mean of 30 measurements of different droplets ondifferent surface regions of each sample � standard deviation.

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was 1.067 L �m�2 �h �mbar, with an R2 value of 0.95

(Figure 4a). The structural characteristics of PESM mem-

branes were responsible for the high water permeance.

The foamy support structure in-between capillaries shows

a permeability that is 1 000 times higher than the filtration

surface so that an equal distribution throughout thewhole

cross section of the fiber is ensured.

Permeation measurements of metabolite solutions

through multibore membranes are reported in Figure 4b.

The convective permeance calculated were: 0.845

L �m�2 �h �mbar (R2¼ 0.997) for albumin solutions, 0.724

L �m�2 �h �mbar (R2¼ 0.993) for IgG solutions and 0.382

L �m�2 �h �mbar (R2¼ 0.93) for dextran solutions. The

average value of the transmembrane fluxes for the

investigated metabolites decreased with increasing

the MW of metabolites. As a result, the flux for BSA

solutions was significantly greater than that for IgG and

dextran.

Concentration profiles of metabolites permeating

through multibore PESM membrane, as monitored in

the extrafiber space and normalized on the feed solution

Figure 4. a) Hydraulic permeation measurements of multiborePESMmembranes. Experimental values (symbols) were averagedon 10 measurements. The interpolation of experimental data isreported as solid line. Slope: 1.067 L �m�2 �h �mbar. b) Convectivepermeation measurements of metabolite solutions through mul-tibore PESM membranes: (~) BSA, (&) IgG, (*) dextran atdifferent transmembrane pressures (DP). Experimental values(symbols) were averaged on 10measurements. The interpolationsof experimental data are reported as solid line. Slopes: 0.845L �m�2 �h �mbar for albumin, 0.724 L �m�2 �h �mbar for IgG and0.382 L �m�2 �h �mbar for dextran.

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Figure 5. Concentration profile appearance of IgG (~) and albu-min (&) in the extrafiber side of multibore membrane module atconstant transmembrane pressure (15 mbar).

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concentration, increased with time and reached a plateau;

this behavior is exemplified in Figure 5. The same trend

was observed at different transmembrane pressure

differences. This characterization provided metabolite

and transport information specific to the membranes,

which should guarantee a sufficient level of mass transfer

for the survival of hepatocytes.

Fluid Dynamics of the Bioreactor

An important characteristic of the membrane bioreactor is

the fluid dynamics characterization under operating

conditions. The bioreactor was connected to a peristaltic

pump that allowed the feeding of nutrients and metabo-

lites and the continuous mixing of the medium inside the

bioreactor. Tracer experiments were performed at an

increasing perfusion flow rate Qin in order to investigate

the bioreactor fluid dynamics. As is shown in Figure 6, for

medium challenge, after a relatively short transience, the

medium concentration in the stream leaving the biore-

actor attained a steady state, then remained constant

Figure 6. Cumulative RTD of the bioreactor following a stepchange of William’s medium concentration in the perfusionmedium. Approach to a steady state of the fractional exit con-centration for Q¼0.48 mL �min�1 (&), Q¼0.64 mL �min�1 (^);(- - - -) tracer concentration stepwise.

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throughout the duration of the experiment and in a time

closely depending on the flow rate Qin. Specifically,

the time needed to attain steady state decreased with

the increasing of the flow rate in the inlet stream. Diffusive

resistances in the intraluminal compartment of fiber

were observed at low flow rate (0.48 mL �min�1), in which

the fractional exit concentration values evidenced a

different concentration between the shell and intralum-

inal compartments. Increasing the flow rate to a value of

0.64 mL �min�1 allowed having the same tracer concen-

tration in both compartments. In order to obtain the same

molecule distribution in the shell and lumen compartment

and an appreciable cell metabolic conversion, a perfusion

flow rate Qin of 0.64 mL �min�1 was chosen. Under these

operating conditions the metabolite concentration in the

intraluminal compartment is uniform and equal to that in

the shell compartment of the bioreactor. By plotting the

RTD according to Equation (2) the mean residence time

was calculated corresponding to a value of 27.5 min. This

value is in agreement with that theoretically predicted

according Equation (3).

Cell Culture in the Multibore Fiber Bioreactor

In the membrane bioreactor we cultured human hepato-

cytes inside the multibore membranes, which is an

experimental system by which human-specific metabolic

information can be studied.

After 48 h of culture the cells on the membranes were

observed by confocal laser microscopy and appeared to be

reorganized in small aggregates. Actin (green staining)was

organized in the cytoskeleton ensuring a round shape to

the cells (Figure 7a–c). A peripheral distribution of vinculin

(red staining) of the hepatocytes evidences the localization

of focal adhesion (white arrows) and a central position of

nucleic acid (blue). The actin is more condensed and

exhibited a circumferential organization which reflects

balanced cell-membrane interactions and cell-cell con-

tacts.[24,25] No stress fibers were developed by the cells. As

the vinculin staining shows, cells developed focal adhesion

contacts, which are the sites of cell attachment to the

substratum that occurs through the link between the

extracellular matrix (ECM) and the molecules of actin

cytoskeleton. The focal adhesions consist of clustered

integrins that are employed, during the steps of cell

adhesion, in physical anchoring processes as well as in

signal transduction through the cell membrane that

influence the maintenance of liver specific functions.[24,25]

A sufficient number of focal adhesions are visible

indicating the sites of interactions with the membrane

surface in different bores of the fiber (Figure 7a–c).

Hepatocytes maintained their morphology with time.

After 14 d of culture they appeared at the SEM analysis

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Figure 7. Confocal images of human hepatocytes in multiborefiber bioreactor after 48 h of culture by actin staining withFITC-phalloidin (green) and vinculin staining with primary anti-vinculin and secondary TRICT antibody conjugated (red) and bynucleic acid stainingwith DAPI (blue). White arrows indicate focaladhesions. (a), (b) and (c) Human hepatocytes in different bores ofthe fibers.

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(Figure 8a, b) to be mostly round in shape and surrounded

by an ECM-like structure. The cells attached onto the

membrane established cell-cell contacts (Figure 8b). The

maintenance of this morphology is important for

the expression of the hepatocyte differentiated functions.

As demonstrated by other studies on hepatocytes cultured

in other hollow fiber membrane bioreactors, limitation

to mass transport of oxygen and nutrients affect

the distribution and viability of cells.[13] Interesting

approaches undertaken by some authors developing

mixing hollow fiber bioreactor with different fibers or

coaxial hollow fiber membrane reactors confirmed the

Figure 8. SEM images of human hepatocytes after 14 d of culturein the lumen of multibore PESM membrane at different magni-fications: (a) distribution of cells inside the fiber lumen; (b) tightjunctions.

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Figure 9. Urea synthesis of human hepatocytes cultured in the multiborefiber bioreactor for 14 d. The values are the mean of six experiments �standard deviation.

678

importance to enhancemass transfer.[6,26] In themultibore

fiber bioreactor, the high permeability of the fiber support

structure and the optimized fluid dynamics conditions

ensure an adequate mass transport throughout the whole

cross section of the fiber with the maintenance of cell

viability.

Functions of Human Hepatocytes in the MultiboreFiber Bioreactor

One of the primary goals of this work was to sustain the

cell-specific functions of the human hepatocytes cultured

in the multibore fiber bioreactor. Specific metabolic

functions in terms of urea synthesis as well as secretion

of proteins were sustained for the investigated culture

time, demonstrating the maintenance of viability of the

hepatocytes cultured in themembrane bioreactor. Figure 9

shows the concentration profile of urea in the shell of the

bioreactor. The urea was produced by cells in the

bioreactor at high levels maintaining their specific activity

for 14 d of culture. The protein secretion by hepatocytes

Figure 10. Protein secretion of human hepatocytes cultured in the multibore fibermembrane bioreactor for 14 d. The values are the mean of six experiments �standard deviation.

was monitored by culturing cells in serum-

free medium (Figure 10). The concentration

of proteins in the shell was maintained for

most of the time at a value of around

150 mg �mL�1. These data take into account

all proteins secreted and released by cells in

the culture medium including the extra-

cellular matrix proteins.

The liver is the most important organ

concerning biotransformation of xenobio-

tics that occurs through the cytochrome

P450s, which is a class of constitutive and

inducible hemoprotein enzymes that meta-

bolize many endogenous substrates as well

as numerous xenobiotics and therapeutic

agents. CYP3A4 is the isoform present in

the highest quantity and is considered to

catalyze the transformation of a large

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number of xenobiotics. The biotransformation

functions of human hepatocytes in the bioreactor

were investigated by using the H. perforatum

extract and paracetamol.

In particular the elimination of hyperforin that is

one of the most abundant lipophilic compounds

and an important active component of St. John’s

wort was evaluated. Human hepatocytes cultured

in the membrane bioreactor were able to biotrans-

form the hyperforin contained in the H. perforatum

present in the medium maintaining a good

metabolic activity for most of the culture time

(Figure 11). The hyperforin biotransformation

occurs via a hydroxylation pathway catalyzed by

CYP3A isoforms. This component contributes also

to the CYP3A4 inducing effect of the H. perforatum

extract. The hyperforin is a potent ligand for the pregnane

X receptor, an orphan nuclear receptor that regulates the

expression of the cytochrome P450 3A4 monooxy-

genase.[27] Treatment of human hepatocytes with hyper-

icum extract results in an induction of CYP3A4 expression,

which is involved in the oxidative metabolism of more

than 50% of all drugs including paracetamol.

Hepatocytes are also able to biotransform acetamino-

phen through the formation of the main metabolite

paracetamidophenyl-b-glucuronide, which is the product

of a glucuronidation reaction (Figure 12). The APAP is

primarily metabolized in the liver by the phase II routes,

sulfation and glucuronidation. In vivo the majority of the

paracetamol is metabolized by glucuronidation which

yields a relatively non-toxic metabolite. A small amount

(approximately 10% of paracetamol dose) of the drug

is metabolized by isoenzyme CYP1A2 and CYP2E1 into

N-acetylbenzoquinoneimine, which is extremely toxic

to liver tissue and it is immediately inactivated by

conjugationwith glutathione.[28] In the human hepatocyte

DOI: 10.1002/mabi.200600281

Human Hepatocyte Morphology and Functions in a Multibore Fiber Bioreactor

Figure 11. Hyperforin concentration in the inlet medium (&) and in outlet medium from bioreactor loaded with cells (&) in presence ofH. perforatum (1.8 mg �mL�1) added to the culture medium.

bioreactor we detected paracetamidophenyl-b-glucuronide

as a product of the glucuronidation of the drug. These results

demonstrated that hepatocytes maintain their biotransfor-

mation functions, which involve the activity of enzymes

catalyzing the conjugation reactions and the CYP1A2,

CYP2E1 and CYP3A, the most active isoforms involved in

the metabolism of hyperforin and acetaminophen.

Conclusion

In summary we report a new membrane bioreactor confi-

guration for the maintenance of human hepatocyte

specific functions. In contrast to the conventional fiber

the PESM multibore membranes have the arrangement

of seven capillaries in one fiber. The multibore fiber bio-

reactor ensures a sufficient oxygenation process, nutrient

feeding, end-product removal and distribution of fluid

molecules inside the cell environment. Human hepato-

Figure 12. Time course of paracetamidophenyl-b-glucuronide for-mation by human hepatocytes cultured in multibore fiber bio-reactor for 14 d in the presence of paracetamol (0.3� 10�3 M).

Macromol. Biosci. 2007, 7, 671–680

� 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

cytes maintained their round shape and showed focal

adhesions as sites of interaction with the membrane

surface. Cells expressed liver specific functions, including

synthetic and detoxification activity for the investigated

culture time as demonstrated by the urea synthesis,

secretion of proteins, the elimination of hyperforin and the

formation of a paracetamol metabolite. These results

demonstrated the feasibility of the multibore fiber

membrane bioreactor that can be used as in vitro liver

tissue model for the evaluation of hepatic metabolic

transformation of compounds and therapeutic molecules

in a well controlled microenvironment.

Acknowledgements: The authors acknowledge the financialsupport of the Italian Ministry of University and Research, MIUR,for the grant to the FIRB research project RBNE012B2K and thefinancial support of the European Commission for the grant to theLivebiomat project STREP NMP3-CT-2005-013653.

Received: December 10, 2006; Revised: January 26, 2007;Accepted: January 31, 2007; DOI: 10.1002/mabi.200600281

Keywords: drug biotransformation; human hepatocytes; mem-brane bioreactor; morphology; synthetic functions

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DOI: 10.1002/mabi.200600281