Telomerase activity and hepatic functions of rat embryonic liverprogenitor cell in nanoscaffold-coated model bioreactor

Shibashish Giri • Karen Nieber • Ali Acikgoz •

Sanja Pavlica • Mario Keller • Augustinus Bader

Received: 14 March 2009 / Accepted: 15 September 2009 / Published online: 9 October 2009

� Springer Science+Business Media, LLC. 2009

Abstract Presently, there is growing interest on telome-

rase activity in all cells (somatic cells, stem cells, cancer-

ous cells and others) since this activity is associated with

cellular changes such as proliferation, differentiation,

immortalization, cell injury and ageing. Telomerase

activity is absent in most of the somatic cells but present in

over 90% of cancerous cells and other immortalized cell

lines. In our present study, we cultured a rat embryonal

liver progenitor cell line RLC-18 in a self-assembly

nanostructured scaffold-coated bioreactor (NCB), colla-

gen-coated plates (CCP) and uncoated plates (UP), and

evaluated changes of telomerase activity by non radioac-

tive techniques (Telo TAGGG Telomerase PCR ELISA,

cell proliferation based on mitochondria number by MTT

assay and hepatic functions such as albumin secretion, urea

metabolism, Cytochrome P450 activity like ethoxyresoru-

fin-O-deethylase (EROD) activity. We found less telome-

rase activity and less cell proliferation, but more hepatic

functions on the NCB than on the CCP and UP. Our data

support the concept that cell-scaffold interaction may play

a significant in controlling the telomerase activity as well

as enhanced hepatic functions. Although our present study

does not focus on the exact mechanism of telomerase

regulation, our result may provide basic clues on cell dif-

ferentiation whereby telomerase activity inhibits differen-

tiation of cells as in the rat embryonic liver cell line, may

be regulated by cell–scaffold interaction and where there is

less proliferation, cells perform enhanced hepatic func-

tions, thereby implying that bioartificial liver support may

be possible.

Keywords Albumin secretion � Bioreactor � Telomerase �RLC-18 cell line � Urea synthesis

Introduction

cCell source is an important component for both basic and

clinical studies and plays a significant role in various organ

support systems including the bioartificial liver support

(BAL), which is an alternative to organ transplantation.

Primary human cells or nonhuman cells are always prefer-

able for all experiments as primary cells are more relevant

to reflect the in vivo situation. Practically, primary cells are

not always easily available, and the use of human embry-

onic cells raises major ethical issues. Therefore, many

researchers use the cell line alternative to primary cells.

Although, it is sometimes difficult to detect the metabolites

of drugs expressing enzymes in cell lines such as HepG2,

BC2 cell lines and other hepatoma cell lines due to the

absence or low expression level of phase I and phase II drug

metabolizing enzymes, it is a quick and effective alternative

model to provide pre-information. In our present investi-

gation, we used the rat embryonal liver progenitor cell line

(RLC-18) [1] as an alternative model to primary human

embryonic liver cells. Although primary adult hepatocytes

are considered the best cell source for BAL, embryonic liver

cells have many advantages over primary hepatocytes for

proliferation in vitro to transplantation in vivo (see review

[2]). This concept led us to design this study by using the

nonhuman embryonic liver cell line.

S. Giri � A. Acikgoz � S. Pavlica � M. Keller � A. Bader (&)

Department of Cell Technologies and Applied Stem Cell

Biology, Biomedical-Biotechnological Center (BBZ),

Deutscher Platz 5, 04103 Leipzig, Germany

e-mail: Augustinus.Bader@bbz.uni-leipzig.de

K. Nieber

Institute of Pharmacy, Pharmacology for Natural Sciences,

University Leipzig, Talstrasse 33, 04103 Leipzig, Germany

123

Mol Cell Biochem (2010) 336:137–149

DOI 10.1007/s11010-009-0266-3

Cell lines now have a very high priority in place of

primary cells in a wide range of biomedical research.

Generally, somatic cells meet cellular senescence after a

few rounds of cell division, and telomerase activity is not

detectable in adult mammal [3]. However, it has been

reported that the ectopic expression of telomerase in pri-

mary somatic cells is sufficient to prevent the telomere

shorting, leading to infinite proliferation, and is therefore

used as a cell line [4]. Immortalized cell lines have more

advantages than primary mature cells such as they have

better uniform cultures, have easier availability, easy

maintenance and easier genetic manipulation, and are more

suitable for drug biotransformation. However, the main

disadvantage is that the telomerase can cooperate with

oncogenes to create a tumourgenic phenotype [5].

Telomerase can inhibit cell differentiation and promote

cell immortality, and telomerase activity is very high in

most tumour cells [6]. Tumour can originate from uncon-

trolled cell proliferation and create cancer by suppressing

apoptosis. It has been reported that telomerase activity is

positive in almost all human cancers originating from

ovary, breast, prostrate, colon, stomach, liver, mammary

glands, brain, etc. [6, 7]. Recent evidence supported the

notion that differentiation of immortal cells inhibits telo-

merase activity [8]. Kaito et al. [9] reported less prolifer-

ation and more hepatic functions of Hep bcl2 without

collagen. It has been shown that telomerase activity

decreases during neuronal differentiation [10, 11]. There-

fore, this investigation indicates that controlling telomerase

activity is essential for the cells to differentiate. We have

proposed here to control the telomerase activity in a

nanoscaffold coated on six-small-well bioreactor with

enhanced hepatic functions.

Bioartificial liver supports are considered as temporary

livers for patients awaiting liver transplantation [12–18].

However, cell source is a major issue, and long-term cul-

ture with enhanced functions relies on a microenvironment,

in particular growth factors, scaffold and bioreactor. The

present study focused on how the nanoscaffold-based

bioreactor enhances the hepatic functions which are one of

the important components in BAL. Based on our previous

studies, our bioreactor enhances oxygenation and provides

a good environment for hepatic function [13, 19–21]. Over

the past few decades, many researchers have focused on

using a scaffold to improve cell function. Conventional

existing scaffolds such as poly(L-lactic acid) (PLLA),

poly(dl-lactide-coglycolide) (PLGA), PLLA–PLGA and

other biomaterials, including alginate and agarose, are in

the micrometre range which did not meet the in vivo

extracellular matrix requirements. Although collagen and

matrigel are widely used as an alternative to the extracel-

lular matrix, these are animal derived and are a major

drawback, in particular, in signalling behaviour.

Furthermore, there may be a chance of contamination and

zoonotic infection in the clinical experiment of animal-

derived materials. The use of a suitable nanoscaffold which

mimics the in vivo situation of hepatocytes for long-term

culture with an enhanced function is needed to overcome

this problem. In order to avoid interference in the signal-

ling behaviours like animal-derived scaffold such as col-

lagen and matrigel, we selected well-characterized

synthetic nanostructured self-assembling scaffold called

PuraMatrixTM.

PuraMatrixTM serves as a synthetic biodegradable alter-

native to animal-derived biomaterials such as collagen and

matrigel. PuraMatrixTM, a synthetic peptide consisting of

a 16-amino acid sequence (AcN-RADARADARADA

RADA-CNH2) which is 99% water content and the

amphillic nature of PuraMatrixTM, gives rise to a sponta-

neous assembly of a water soluble beta sheet structure in the

presence of monovalent cations [22, 23]. The fibre and pore

sizes of PuraMatrixTM are 10 and 5–200 nm, respectively,

which are similar to those of the in vivo extracellular matrix

[22–24]. Recently, there has been much evidence in a wide

range of cells for differentiation by using PuraMatrixTM.

For example, for the nerve [25–28], cartilage [29–31], liver

[32, 33], cardiomyocyte [34, 35] and vascular endothelial

cells [34–37]. This scaffold facilitates bone regeneration in

bone defects of calvaria in mice [38] and accelerates wound

healing [39]. It is considered as best model for biofunctional

[40]. Recently, it has been reported that mesenchymal stem

cells can differentiate into mature osteoblasts to form

mineralized matrices with this peptide hydrogel [41]. Fur-

thermore, investigations have shown that PuraMatrixTM can

be a promising novel scaffold candidate to examine ECM-

based signalling [42] overcoming the regular problems

associated with existing standard scaffolds such as matrigel

and collagen. Little is known about the control of telome-

rase activity particularly in nanoscaffold–cell line interac-

tion in an appropriate condition like culture in small scale

bioreactor.

In our present study, we analysed the telomerase activity

by TeloTAGGG telomerase PCR ELISA Kit which entails

nonradioactive techniques and provides a way to perform a

highly sensitive enzyme immunoassay for the detection of

telomerase. We used simple methods colorimetric assay

(MTT based) to investigate the cell number based on

mitochondria metabolic activity. It is also the non-radio-

active quantification of cell proliferation and viability.

Finally, we showed the attachment profile by using trypan

blue exclusion. We used the six-small-well bioreactor to

evaluate the telomerase activity along with the hepatic

function (albumin secretion, urea metabolism and P450

cytochrome activity). We cultured RLC-18 cell lines in the

NCB, CCP and UP to show the telomerase activity and

hepatic functions.

138 Mol Cell Biochem (2010) 336:137–149

123

Materials and methods

Materials and chemicals

All chemicals used in this study were of the highest purity.

Collagenase, penicillin and streptomycin were obtained

from Biochrom, Berlin, Germany, and all other cell culture

reagents were purchased from Gibco, Weinheim, Germany.

The other remaining chemicals were obtained from Carl

Roth GmbH (Karlsruhe, Germany) and Sigma-Aldrich

(Munich, Germany). PuraMatrixTM purchased from BD

science, MA. TeloTAGGG Telomerase PCR ELISA kit

(Cat no. 11854666910) obtained from Roche Diagnostics

GmBH, Mannheim, Germany.

Preparation of CCP and NCB

Rat tail collagen was prepared according to the method

[43]; a concentration of 1.5 mg/ml collagen was used for

six-normal-well plates to create a thin layer of collagen gel

as a bed for cell lines. First, we decreased the viscosity of

the PuraMatrixTM stock solution of our aliquots (1.5 ml

microtube) by vortexing 30 min in a bath sonicator. If air

bubbles were present, we centrifuged the aliquots at high

speed for a few seconds. We prepared 0.25% of the

PuraMatrixTM by diluting with sterile water. In order to

create nanoscaffold in the six-well plate bioreactor, 1.2 ml

of 0.25% (v\v) PuraMatrixTM was uniformly distributed

over each well and then 2.4 ml of RPMI 1640 medium was

added to each well very carefully. In order to promote the

gelation, we put the bioreactor in an incubator for 1 h.

After the nanostructure hydrogel was assembled, we care-

fully changed the medium with a wide top micropipette.

We avoided using the aspirator because of the greater risk

of destroying the nanostructured hydrogel. We changed the

medium (300 ll per well for 24 wells) twice over a period

of 1 h to equilibrate the physiological pH and finally put it

in an incubator overnight with the medium. Over the next

few days, we used the nanoscaffold-coated bioreactor

(NCB) for the present experiment. 100,000 cells were

seeded in each well. The culture medium (250 ll) was

replaced with fresh medium in every 24 h, and the total

amount of supernatant (200 ll) was collected for LDH,

albumin, Urea test, EROD activity in every day.

Culture of the RLC-18 cell line

The cell line RLC-18 (17 days embryonic liver of Japanese

albino rat) was obtained from the German Collection of

Microorganisms and Cell Cultures (DSMZ, Braunschweig).

RLC-18 was cultured in six UP, CCP and the NCB in RPMI

1640 medium supplemented with 5% FCS, dexamethasone

(0.7 lg 9 ml-1), gentamycin (50 lg 9 ml-1), L-glutamine

(292 lg 9 ml-1), glucagon (0.1 lg 9 ml-1) and insulin

(10 lg 9 ml-1) at 37�C in a humidified atmosphere con-

taining 5% CO2.

Bioreactor model

The model has been explained in detail in a previous study

[13, 44, 45]. In brief, it is a modified form of a conventional

six-well cell cultivation plate and is composed of a scaffold

of polycarbonate with six wells, the gas-permeable PTFE

membrane of 25 lm thickness, a six-hole silicon seal and a

six-hole metal base (Fig. 1). The PTFE membrane is a

transparent, thermoplastic film with the oxygen perme-

ability of 114.5 cm3 m-2 24 h-1 k Pa-1 and allows a

maximal oxygen supply of 90 mmol per well (1.77 cm2)

per day for cell cultures. Based on this model, the amount

of oxygen required by the hepatocytes (2.5 9 105 cells/

well) in the bioreactor is suggested to range between 6.5

and 19.5 mmol, allowing a 5–14 times higher oxygen

supply than needed. The bioreactor was placed into which

admitted a direct delivery of oxygen to the cells from the

bottom of the device. It has been reported that in vitro

hepatocyte culture exhibits more oxygen uptake than other

cells [46]. It enhanced oxygenation to in vitro cell culture

of hepatocyte, supports to maintain stable liver specific

Fig. 1 The small-scale

bioreactor is composed of a

polycarbonate scaffold with six

wells, a gas-permeable PTFE

membrane, a six-hole silicon

seal and a six-hole metal base.

96-well bioreactor is same like

six-well bioreactor except

96-well, 96-hole silicon and

96-hole metal base. Enhanced

oxygen to the cells from the

bottom of bioreactor

Mol Cell Biochem (2010) 336:137–149 139

123

functions since hepatocyte perform more than 500 func-

tions of liver. Conventional methods rarely focused for

adequate supply of oxygen for hepatocytes. We used our

six-well bioreactor to over come this limitation. For anal-

ysis of BrdU incorporation, we used PuraMatrixTM coated

96-well bioreactor in place six-well bioreactor and com-

pare with CCP and UP.

Telomerase activity test

Telomerase activity was determined using the Telo TAG-

GG Telomerase PCR ELISA kit (Roche, Mannheim,

Germany), which is based on the Telomeric Repeat

Amplification Protocol assay with a nonradioactive ELISA

detection. The procedure was performed using 200,000

cells per single reaction of RlC-18 cells in six UP, CCP and

NCB in accordance with the manufacturer’s instructions.

MTT assay

Cell proliferation and viability assays are of particular

importance for routine applications by MTT assay. Tetra-

zolium salts (e.g. MTT) are especially useful for assaying

the quantification of viable cell, because they are cleaved to

form a formazan dye only by metabolically active cells.

MTT was dissolved at a concentration of 5 mg per ml in

PBS (pH 7.4) and filtered for sterility. We washed once

with PBS and added 1 ml of MTT stock solution and

incubated at 37�C for 2 h. Then we added the same volume

(1 ml) of lysis buffer to each well and shaken 10 min for

500–800 rpm. After shaking, the optical density was

immediately measured. The optical density of each well

was measured using an automatic micro-plate reader

(Tecan, Switzerland) with a 630-nm reference wavelength

and 570 nm test wavelength. The MTT assay is a state of

mitochondria activity, but when the power house (mito-

chondria) failure is occurred, liver failure is occurred [47].

BrdU proliferation test

Cell proliferation was assessed by counting the cells and by

detecting the incorporation of 5-bromo-29-deoxyuridine

(BrdU) into the DNA in three independent experiments. For

the BrdU incorporation assay, we used the same number of

cells. After its incorporation into the DNA, BrdU was

detected by an immunoassay using a BrdU incorporation

assay kit according to the manufacturer’s instructions

(Roche, Mannheim, Germany). The reaction product was

quantified by measuring the absorbance using an ELISA

reader at 450 nm (reference wavelength 690 nm). The same

procedure was performed for negative control samples

which were identical but without BrdU labelling.

Trypan blue exclusion

The viability of the RLC-18 cell line of three plates

including the bioreactor was determined by trypan blue

exclusion analysis. An equal volume of trypan blue reagent

was added to a cell suspension and the percentage of viable

cells was evaluated under the field microscope. Assays

were performed in triplicate.

LDH test

Lactate dehydrogenase activity (LDH) was measured based

on our previous report [44]. In the first step, NAD? was

reduced by the LDH-catalysed conversion of lactate to

pyruvate. In the second step, the catalyst transferred H/H?

from NADH/H? to a tetrazolium salt which is reduced to

the coloured formazan. This product was then measured at

490 versus 600 nm in the photometer. Calibration was

performed with L-LDH standards.

Albumin and urea test

Albumin production by RLC-18 cell line in culture was

measured in supernatant medium collected every other day

using our previous described ELISA method [48]. Briefly,

albumin secretion was determined immunochemically with

ELISA technique [49], using culture supernatants were

collected every 24 h up to 7 days in culture and stored at

4�C for further analysis. Chromatographically purified rat

albumin and the monoclonal antibody for rat albumin were

purchased from Cappel (Durham, NC). 96 well plates

(Nunc, Wiesbaden, Germany) were coated with albumin

and left overnight at 4�C. The coating buffer contained

1 mg/ml albumin [49]. After washing the plate four times,

50 ll of cell culture supernatant was added to the wells and

incubated with 5 ll of anti-rat albumin antibody conju-

gated to horseradish peroxidase. After 24 h at 4�C,

substrate buffer containing O-phenylenediamine dihydro-

chloride and H2O2 was added for 6 min. The reaction was

stopped with 100 ml of 8 N H2SO4. Absorbance was

measured at 490 nm using a Tecan spectrophotometer. The

standard curve ranged between 1 and 100 mg albumin/ml.

Urea secretion studies

Urea concentration was assayed by the enzymatic urease

method (Sigma). Cell culture supernatants were collected

every 24 h up to 7 days in culture. The experiments were

repeated thrice with cells from three different isolations.

Data are shown in millimole according to international SI

classifications.

140 Mol Cell Biochem (2010) 336:137–149

123

EROD activity

Ethoxyresorufin-O-deethylase (EROD) activity is a bio-

marker of chemical exposure involving a cytochrome P450

function which is mainly catalyzed by isoenzymes

CYP4501. In order to evaluate the EROD activity, cells

were seeded in all plates including the six-well plate NCB

at a concentration of 1 9 105 cells/well and incubated for

24, 48, 72 and 120 h. Deethylation of ethoxyresorufin is

associated with CYP 1A. As described before [50], hepa-

tocyte cultures were each incubated with 10 lM of

7-ethoxyresorufin and dicumarol. Dicumarol was added to

the assay media to prevent further biotransformation of

resorufin by cytosolic diaphorase. Aliquots of the super-

natant medium were withdrawn after 1 h of incubation.

Samples were stored at -20�C until analysis. After

thawing, resorufin conjugates were cleaved using

b-glucuronidase in 100 U/ml acetate buffer overnight at

37�C. Aliquots of the so-treated samples were mixed with

glycine buffer (1.6 M, pH 10.3). Afterwards, formation of

resorufin was quantified by fluorometry with an excitation

wavelength of 530 nm and an emission wavelength of

580 nm. The spectrofluorometer was calibrated using

resorufin standards.

Statistical analyses

The experiments were independently repeated 3–5 times,

and data are expressed as mean ± SD. The results were

tested for statistical significance with the Student’s t test.

P values less than 0.05 were considered to be statistically.

Results

Phase construct observation

The design of the bioreactor allowed the observation of the

state of RLC-18 cell lines with an inverse microscope.

Hepatocytes cultured in the bioreactor adopted an in vivo

like polygonal and established extensive cell-to-cell con-

tracts with natural organization. There is no significant

dissimilarity on days 1 and 2 in all three plates including

the NCB (Fig. 2). However, on day 3, RLC-18 cell lines

seemed to be better organized with well-developed

polygonal shapes in the NCB than in the CCP and UP

(Fig. 2). Hepatocytes cultured in the bioreactor adopted an

in vivo like polygonal shape and established extensive cell-

to-cell contracts with natural reorganization. Optical images

Fig. 2 Phase constract images

of RLC-18 cell lines cultured in

uncoated six-well plates,

collagen-coated six-well plates

and nanoscaffold coated six-

well Bioreactor. Three images

of first lines of day 1 and other

two lines of images of days 2

and 3

Mol Cell Biochem (2010) 336:137–149 141

123

of 3 days culture of RLC-18 cell lines in conventional

collagen-coated plates and uncoated plates are hypothe-

sized to assist in enabling a significant increase in prolif-

eration of RLC-18 cell lines continuously and received

apoptosis due to high confluence. It has been reported that

apoptotic death occurred when cell line grown to conflu-

ency [51]. For example, rat embryo fibroblast cells are

immortalized by viral transfection for infinite proliferation.

These cells have been observed to die via an apoptotic

death when grown to confluency [52]. However, NCB

inhibited continuous proliferation and enhanced liver

function are discussed later in this present study.

Telomerase activity

We analysed the telomerase activity from day 1 to day 3 in

the CCP, UP and NCB (Fig. 3). Telomerase activity was

detected from day 1 to day 3 by most sensitive telomerase

ELISA–PCR method. We found telomerase activity to be

almost the same in CCP and NCB initially on day 1, but

significantly different with uncoated plates. Interestingly,

we detected twice the amount of activity in the UP and

CCP than in the bioreactor on day 2. The telomerase

activity is almost the same on day 2 in UP and CCP. It is

believed that during continuous cell proliferation of cell

lines, cells may not perform enhanced functions. On day 3,

telomerase activity is significantly increased up to nearly

twofold in UP and CCP. The telomerase activity is much

higher than in the positive control in all cases including

NCB because RLC-18 is also an immortalization-based

cell line. Generally, immortalization-based cell lines show

strong telomerase activity. The present consideration based

on 3-day basic analysis of telomerase activity indicates that

it may be possible to control some extent of telomerase

activity under certain condition like culture NCB. We are

in process to further control telomerase activity of this cell

line by adding suitable agents (data not shown).

MTT assay

The MTT assay is a standard colorimetric assay (an assay

which measures changes in colour) for measuring the

activity of enzymes that reduce MTT to formazan, giving a

purple colour by the action of dehydrogenase enzymes

which are reproduced by living cells. This mostly happens

in mitochondria, and then, the assays are therefore largely a

measure of mitochondrial activity. These reductions take

place only when mitochondrial reductase enzymes are

active, and therefore, conversion is often used as a measure

of viable (living) cells which indicates living cell prolifer-

ation activity. We measured the MTT assay everyday up to

3 days in UP, CCP and the NCB (Fig. 4). We found one to

twofold more cell proliferation in UP and CCP in the first

3 days. In the NCB, there was a threefold less proliferation

than in the CCP on the first 3 days. From day 1, the cell

proliferation is significantly different up to day 3. There-

fore, the RLC-18 cell line stimulates proliferation more in

CCP and UP. Taken together, we found less cell prolifera-

tion in the NCB from day 1 to day 3 when compared to CCP

and UP. The cultural protocol is previously described [44].

Fig. 3 Telomerase activity based on telomerase signal (Optical

density, OD) in 3 days of culture. Results are presented as the

means ± SD from three independent experiments. Statistically sig-

nificant difference compared with controls (P \ 0.05)

Fig. 4 Cell proliferation (percentage) test based on MTT test.

Results are presented as the means ± SD from three independent

experiments. Statistically significant difference compared with con-

trols (P \ 0.05)

142 Mol Cell Biochem (2010) 336:137–149

123

BrdU proliferation test

This cell proliferation by ELISA is a fast, simple colori-

metric method, and an alternative to quantitative cell pro-

liferation based on the measurement of BrdU incorporation

during DNA synthesis. We analysed RLC cell proliferation

based on the measurement of BrdU incorporation in newly

synthesized cellular DNA. We cultured 1,000 cells per well

in a 96-well NCB, CCP and UP and showed BrdU prolif-

eration in an ELISA assay in a time period of 6, 12 and

24 h (Fig. 5). This 96-well bioreactor is the same as the

six-well bioreactor as described above (Fig. 2c). In the first

6 and 12 h, BrdU proliferation of CCP is onefold higher

than UP and NCB. In fact, the proliferation in CCP was

higher only after 12 h, after 6 or 24 h, than the others

(Fig. 5). However, in the NCB, it is always constant except

for a slight increase after 24 h. However, in the NCB, it is

constant or it slightly differs on first 2 h and had increased

value of BrdU incorporation on 24 h. This result also

supports the notion that NCB inhibits the cell proliferation.

Trypan blue exclusion test

The dye exclusion test is used to determine the number of

viable cells present in a cell suspension. We analysed the

cell viability for the same condition, such as telomerage

activity, to correlate the telomerase activity data from day 1

to day 3 (Fig. 6). The influence of the nanoscaffold on cell

viability during proliferation has also been assessed by the

Trypan exclusion method and compared with the CCP and

UP. We found almost the same number of viable cells on

day 1, with slightly different viability. We showed a two-

fold increase in viable cells in the CCP and UP. The

number of viable cells is also higher in the CCP than in the

NCB on day 2. Interestingly, from a vitality point of view,

the cells of the bioreactor always show 95% viability

throughout the culture period up to day 3. Although the cell

number of CCP and UP is somehow higher there is always

less viability than in the bioreactor. We found that of the

total cells, 70–80% were viable out of total cell in CCP and

UP, and this was 90% in NCB culture. Therefore, this may

be apoptosis due to continuous proliferation. However, in

the case of the NCB, this inhibited proliferation and has a

higher vitality percentage of cells when compared with the

other two plates. The doubling time of RLC-18 cell line is

48–72 h. Cell line grown to confluency is associated with

apoptotic cell death was observed in 13 of 14 rodent and

human cell lines [51]. Tumour cells not only exhibit

excessive proliferation, but also undergo apoptosis at rates

that far exceed those in normal tissue [52]. Herein, culture

in the NCB significantly prevented cell death during pro-

liferation. These results could also indicate that nanoscaf-

fold inhibited cell proliferation, with high vitality for

enhanced functions. Taken altogether, we showed the

suppressed proliferation in NCB culture had cell popula-

tion with higher number of viable cells.

LDH test

The cellular integrity of the RLC-18 cell lines in the con-

ventional CCP and UP and in the NCB over the cultivation

period of 7 days was examined every day by their release

of LDH into the supernatant of the medium. LDH content

not significantly different in all plates including the NCB

Fig. 5 BrdU corporation of 3 days of culture. Results are presented

as the means ± SD from three independent experiments. Statistically

significant difference compared with controls (P \ 0.05)

Fig. 6 Viable cells were counted by the trypan blue exclusion

method with the use of a hemocytometer. Results are presented as the

means ± SD from three independent experiments. Statistically sig-

nificant difference compared with controls (P \ 0.05)

Mol Cell Biochem (2010) 336:137–149 143

123

on day 1. However, it was much higher in CCP and UP

since from day 3 to 7 than of NCB. The later one was not

only lower but also nearly unaltered throughout the period

of experimentation (Fig. 7). This is an indication that there

is no cell membrane integrity in NBC. We hypothesize that

there is much proliferation and finally causing apoptosis

due to the lack of space with continuous proliferation. It

has been reported that cell death occurred in immortalized

and non-immortalized cells at confluency [51]. These

findings demonstrate that the bioreactor has major advan-

tages over the conventional collagen-coated plates

regarding the cellular integrity.

Synthetic potential of albumin secretion and urea

synthesis

The efficacy of the NCB to support hepatocyte-specific

functionality was evaluated and compared with that of the

conventional CCP and UP by assessing albumin and urea

synthesis by the RLC-18 cell line over the entire cultivation

period of up to 7 days. In two conditions (CCP and UP),

the ability of RLC-18 cell lines to produce albumin is

almost same through 7 days of culture and the albumin

secretion between these conditions is one to twofold higher

in CCP and UP. In the case of the NCB, starting on day 1

the albumin secretion was already twofold higher then

CCP, and then significantly increased on days 2 and 3.

Then the albumin secretion slightly decreased on day 4 to

day 6. High rates of albumin synthesis for the cells in the

plates were obtained on days 3 and 7 (Fig. 8). Levels of

albumin secretion in the NCB appeared to remain stable

from day 2 to day 7 until the end of the cultivation period.

Urea synthesis was analysed as a parameter for detoxi-

fication potential in 7 days of culture in all plates (Fig. 9)

like albumin secretion. The rate of urea synthesis in the

bioreactor increased from day 1 to day 6 and decreased on

day 7 until the end of the cultivation period. By comparison,

Fig. 7 LDH release by RLC cell lines as a parameter for the cellular

integrity. Results are presented as the means ± SD from three

independent experiments. Statistically significant difference com-

pared with controls (P \ 0.05). Each enzymatic activity assay for

1 9 105 cells was done in triplicate

Fig. 8 Albumin Secretion in continuous culture of 7 days. Results

are presented as the means ± SD from three independent experi-

ments. Statistically significant difference compared with controls

(P \ 0.05)

Fig. 9 Urea synthesis in continuous culture of 7 days. Results are

presented as the means ± SD from three independent experiments.

Statistically significant difference compared with controls (P \ 0.05)

144 Mol Cell Biochem (2010) 336:137–149

123

the rates of urea synthesis in the NCB were about threefold

higher than those in the plates (CCP and UP), demonstrating

a two evidences of this oxygen-dependent process by the

use of an oxygenating surface in the bioreactor and scaffold

in nano range like ECM in vivo.

EROD activity (with inducer and without inducer)

In addition, we analysed the EROD activity with inducer

and without inducers because measurement of ethoxyres-

orufin-O-deethylase (EROD) activity in liver cells is a

well-established in vivo biomarker of the exposure to a

wide range of synthetic chemicals or xenobiotc chemicals.

EROD is a highly sensitive indicator of liver toxicity,

providing evidence of receptor-mediated induction of

cytochrome P450-dependant monooxygenases (the CYP1A

subfamily specifically) by xenobiotic chemicals. The

EROD activity of all three systems (NCB, CCP and UP)

with inducer (3 methycholanthrene) and without inducer up

to a 4-day culture period is shown in Figs. 10 and 11,

respectively. The EROD activity in UP is distinctly lower

than in CCP and NCB from day 1 to day 4. Initially, the

EROD activity is slightly higher in the NCB than in the

CCP and continued so to day 2. From day 3, the EROD

activity increased with high activity on day 4. We found

more EROD activity when we exposed with inducer

(3 methycholanthrene) than without inducer. The EROD

activity of the CCP with inducer is only higher on day 2

than in the NCB and remained less in all other conditions.

These results also suggest that the nanoscaffold stimulates

cytochrome P450 activity.

Discussion

Presently, telomerase activity is an active area of research

for both basic and clinical biomedical research. Due to the

irregularity of fresh human samples, many researchers used

to study their experiments by using cell lines as an alter-

native to primary human cells. In most cases, immortali-

zation of somatic cells or other cells based on the recent

advances in telomerase biology and oncogenes led to

unlimited population doubling, which may be a possible

source for all extramural support programs including

BALs. Herein, we focus more on BALs. The performance

of extraporeal liver supports strictly relies on the cell

source and scaffold. Primary human cells are not easily

available, so researchers focused on using cell lines like

HepG2 cell lines and other hepatoma cell lines. The pos-

sible tumourgenic situation in the host is the main disad-

vantage due to the expression of telomerase of cell lines

which stimulates the specific oncogenes of the host tissue

[5]. Telomerase activity has been detected in a number of

human cancers [6, 53, 54] where 90% of all human cancers

contain telomerase activity. Furthermore, it has been

reported that telomerase activity is not always detectable in

immortal cell lines [55]. It has been demonstrated that

treatment with antisense oligonucleotides can inhibit

growth and survival of cancer cells by inhibiting telome-

rase [6, 56], suggesting a control of telomerase activity in

the immortal phenotype. Further neural cells differentiate

into neurons when telomerase activity decreases [6, 11].

Therefore, telomerase interferes with the different potential

of cell. There is an urgent need to determine the regulatory

Fig. 10 EROD activity without Inducers (3 Methycholanthrene) on

4 days of culture. Results are presented as the means ± SD from

three independent experiments. Statistically significant difference

compared with controls (P \ 0.05)

Fig. 11 EROD activity with Inducer (3 Methycholanthrene) on

4 days of culture. Results are presented as the means ± SD from

three independent experiments. Statistically significant difference

compared with controls (P \ 0.05)

Mol Cell Biochem (2010) 336:137–149 145

123

control pathways of telomerase activity and the clinical

diagnosis, prognosis, treatment of cancer and extramural

support like BAL will be important for the differentiation

of many cells.

Malignant cell lines which are currently available

include HepG2 or C3A cells, [57], or xenogenic (porcine)

hepatocytes for BAL, but these carry potential risks to

recipients, e.g., inoculation of tumour cells [58]. This sit-

uation stimulates the generation of a concept to design this

study to control the telomerase activity by nanoscaffold–

cell lines interaction in a small-scale bioreactor to reduce

the chance of creating tumourgenic cells in the host tissue.

In this present experiment, we have shown the basic

information how the NCB interferes with the telomerase

activity and enhances the hepatic functions (albumin

secretion, urea metabolism, cytochrome P450 (EROD

assay). We noticed less telomerase activity in the NCB

than in the CCP and UP. Therefore, this result provides

some information that scaffold–cell interaction is also a

way to control the telomerase activity. Telomerase activity

varies from cell line to cell line, but we used here a 17-day

embryonic liver cell line as an alternative to human

embryonic liver cell because it was embryonic. The main

limitation of foetal or embryonic liver cells is a major

ethical issue but foetal hepatocytes have several advantages

over adult cells. They offer a potentially unlimited source

of cells for hepatocyte replacement [59], whereas primary

adult liver cells are limited due to no further proliferation.

It has been reported that foetal liver progenitor cells have

the potential to continue to proliferate for up to 6 months

after transplantation but adult hepatocytes ceased prolif-

eration within the first month [60, 61].

We selected to use the nano range scaffold PuraMa-

trixTM which can meet the gap of limitation of conven-

tionally existing micrometre range scaffolds. Further, it has

been believed that the scaffold should be smaller than the

cell, so that the scaffold can hold up the cell like in vivo

and maintain prolonged functions without failing.

Animal-derived scaffolds often provide an environment

more similar to the natural ECM than synthetic scaffolds;

one potential problem with all animal-derived biomaterials

is that they can potentially carry dangerous pathogens [62].

Although collagen and matrigels are capable of forming an

environment much more similar to natural ECM but we

selected the self-assembling peptide scaffolds (PuraMa-

trixTM) for this experiment which is not biologically derived

and contains only known constituents which are completely

defined and can be modified without difficulty [63]. Fur-

thermore, PuraMatrixTM neither elicit a noticeable immune

response nor inflammatory reaction in animals, the degra-

ded products can be reused by the body or eliminated easily

from body and may be also be useful as a bio-reabsorbable

scaffold to repair organs [64, 65]. Many synthetic scaffolds

used in tissue engineering release harmful degradation

products, but this nanoscaffold is easily degradable with any

harmful surrounding tissue as 99% is water content.

Quantitatively, the rates of cell replication and of

apoptosis during the development and regression of liver

cancer have been reported [52]. According to their study,

apoptosis rate per day is 1 of 100 normal cells and 9 of 100

tumour cells, termination of cell replication and doubling

as observed after NAF (nafenopin: a liver mitogen) with-

drawal, would follow a daily cell loss rate of 2 for 100

normal cells but 18 for 100 tumour cells. Apoptotic cell

death due to high confluency of high density was observed

in 13 of 14 rodent and human cell lines [51]. It is very

essential to make a proper balance between cell prolifera-

tion and apoptosis for the development and maintenance of

normal organs. Otherwise more chance of occurrence of

cancer due to the smother of normal apoptosis process

which caused more imbalance between cell proliferation

and apoptosis [66].

Animal-derived biomaterials such as bovine collagen

and gelatin, fibronectin, intestinal submucosa, cadaver tis-

sue and matrigel may help to create the 3-dimensional

microenvironments, but complex research and therapies

due to their potential risk of other unknown material con-

taminations, thus rise issues about cell signalling, protein

content and reproducibility. We used PuraMatrixTM

because of its synthetic nature and extreme purity of a

single peptide component. It has potential in many applied

clinical application in coating or to encapsulate cells, tai-

lor-made for particular cells, tissues and therapies. The

peptide sequence of PuraMatrixTM promotes wide range of

cell attachment, but does not mediate argenin–glycin–

aspartic acid (RGD)-dependent integrin signalling since the

peptide sequence of PuraMatrixTM Peptide Hydrogel is

similar to RGD. It remains unclear that whether this syn-

thetic peptide interacts with the hepatocytes integrins to

transducer the signalling for growth or apoptosis in adverse

climate of BAL and a signalling pathway mediated by

extracellular regulated kinase (ERK) pathway. However,

PuraMatrixTM is promoting the cell adhesion and growth.

In some cases in NCB, we found that lower viability in

some cases may be because of less percentage of FCS. As

the RLC-18 cell is an immortalized cell line, still we found

70–80% viability compared to 95% viability in NCB. The

fundamental concept is that NCB provide direct enhanced

oxygenation since hepatocytes were located at a distance of

10–20 lm from oxygen permeable membrane in a mono-

layer formation [67]. CCP and UP do not provide direct

adequate direct oxygenation like NCB. Further research is

needed to add suitable combination of growth factors to

enhance the liver specific functions. The main disadvan-

tages of this study are that using of 5% foetal calf serum,

which also introduces a variety of known and unknown

146 Mol Cell Biochem (2010) 336:137–149

123

promoters and inhibitors. After growing the cells in UP,

CCP and NCB, we tested albumin secretion rate, one of

most important clinical parameter has been used in BAL.

Although the secretion potential of growing RLC-18 cell in

NCB is not same corroborative to the in vivo value, but we

previously reported a BAL system using primary hepato-

cytes and flat membrane bioreactor (same fundamental

basic design concept of NCB) and which was based a

preclinical study in a new porcine hepatectomy model [68].

Every cell line is associated with telomerase biology.

Control of proliferation is an important objective in cell

lines for both the enhanced function and differentiation

mechanism. Generally, it is accepted that during continu-

ous proliferation, cells may not perform their functions

properly. It has been reported that cells proliferate less and

function more in uncoated collagen conditions than colla-

gen-coated conditions [9]. Further investigation is needed

to show the role of many other factors which have the

potential to inhibit the telomerase activity. Telomerase

plays an important role in cellular ageing for infinite pro-

liferation and represents the greatest challenge in regen-

erative medicine and tissue engineering. Data had

suggested that such a functional nanoscaffold could mimic

the in vivo microenvironment and promote cell function by

controlling the telomerase activity, thereby enhancing the

functions of BAL.

In conclusion, our present study provides basic clues

that telomerase activity may be controlled under certain

conditions like our nanoscaffold-coated bioreactor. We

found enhanced albumin secretion, urea metabolism and

higher expression in the EROD assay in a NCB than in

conventional CCB and UP. The main advantages include

the use of nanoscaffold called PuraMatrixTM, like ECM

which is not of animal-derived origin, may be implemented

in extracorporeal bioartificial liver devices without zoo-

notic risk. Taken all together, we reported suppressed

proliferation of rat embryonic liver progenitor cell in NCB

culture had cell population with high number of viable cells

with enhanced liver specific functions. This well-charac-

terized nanoscaffold provides a better physiological sub-

strate for stable but for dynamic cell culture and suggests

its further application for biomedical research, cancer

biology and regenerative biology.

Acknowledgements The authors would like to thank Angela Hen-

ning for technical assistance. This work was supported by LIVEBI-

OMAT (EU project NMP-013653).

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