Maintenance of Hepatic Functions in Primary Human Hepatocytes Cultured on Xeno-Free and Chemical Defined Human Recombinant Laminins
Maintenance of Hepatic Functions in Primary Human Hepatocytes Cultured on Xeno-Free and Chemical Defined Human Recombinant Laminins
Masaaki Watanabe 0 1
Helen Zemack 0 1
Helene Johansson 0 1
Louise Hagbard 1
Carl Jorns 0 1
Meng Li 0 1
Ewa Ellis 0 1
0 Department of Clinical Science, Intervention and Technology (CLINTEC), Division of Transplantation Surgery, Karolinska Institutet, Stockholm, Sweden , 2 BioLamina AB, Stockholm , Sweden
1 Editor: Xuefeng Liu, Georgetown University , UNITED STATES
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Funding: The funder BioLamina AB provided
support in the form of salaries for author LH, but did
not have any additional role in the study design, data
collection and analysis, decision to publish, or
preparation of the manuscript. This does not alter our
adherence to PLOS ONE policies on sharing data
and materials. The specific role of the author is
articulated in the ‘author contributions’ section.
Refined methods for maintaining specific functions of isolated hepatocytes under xeno-free
and chemical defined conditions is of great importance for the development of hepatocyte
research and regenerative therapy. Laminins, a large family of heterotrimeric basement
membrane adhesion proteins, are highly cell and tissue type specific components of the
extracellular matrix and strongly influence the behavior and function of associated cells
and/or tissues. However, detailed biological functions of many laminin isoforms are still to
be evaluated. In this study, we determined the distribution of laminin isoforms in human liver
tissue and isolated primary human hepatocytes by western blot analysis, and investigated
the efficacy of different human recombinant laminin isoforms on hepatic functions during
culture. Protein expressions of laminin-chain α2, α3, α4, β1, β3, γ1, and γ2 were detected
in both isolated human hepatocytes and liver tissue. No α1 and α5 expression could be
detected in liver tissue or hepatocytes. Hepatocytes were isolated from five different
individual livers, and cultured on human recombinant laminin isoforms -111, -211, -221, -332,
-411, -421, -511, and -521 (Biolamina AB), matrigel (extracted from
Engelbreth-HolmSwarm sarcoma), or collagen type IV (Collagen). Hepatocytes cultured on laminin showed
characteristic hexagonal shape in a flat cell monolayer. Viability, double stranded DNA
concentration, and Ki67 expression for hepatocytes cultured for six days on laminin were
comparable to those cultured on EHS and Collagen. Hepatocytes cultured on laminin also
displayed production of human albumin, alpha-1-antitrypsin, bile acids, and gene
expression of liver-enriched factors, such as hepatocyte nuclear factor 4 alpha,
glucose-6-phosphate, cytochrome P450 3A4, and multidrug resistance-associated protein 2. We conclude
that all forms of human recombinant laminin tested maintain cell viability and liver-specific
functions of primary human hepatocytes, and that recombinant laminin is a promising
xenofree and chemical defined strategy for preservation of hepatocyte specific function in vitro.
Competing Interests: The authors have declared
that no competing interests exist.
During the cell isolating process, hepatocytes are separated from each other and deprived of
their original environment [
]. The disruption of cell-to-cell and cell-to-matrix contacts leads
to subsequent hepatocyte dysfunction and/or apoptosis, and the isolated hepatocytes lose their
specific functions if the cells are not anchored and cultured under appropriate conditions [
]. The extracellular matrix contains different specific proteins, such as laminins, type IV
collagens, perlecan, and nidogens, forming complex networks that strongly influence the behavior
and functions of the associated cells . To maintain specific functions in a culture system,
isolated hepatocytes need to be cultured with an extracellular matrix component, usually
Matrigel™ or collagen. However, due to diversity of biological material, the effect on the cells differs
with different matrices. The quality may also differ from batch to batch, making reproducibility
difficult. Furthermore, clinical application under good manufacturing practice (GMP)
protocols is difficult due to their animal origin. A robust and new strategy for culturing isolated
hepatocytes under xeno-free and chemical defined conditions is important for future use of
primary and/or stem cell derived hepatocytes in GMP settings.
Laminins are a large family of conserved, heterotrimeric basement membrane adhesion
proteins that consisting of α-, β-, and γ-chains. Laminins contribute to the structure of
extracellular matrix and influence the behavior of associated cells, such as adhesion, migration,
differentiation, viability and phenotypic stability [
]. In vertebrates, there are at least 16 different
laminin isoforms present, and they are named based on their chain composition, for example,
the composition of laminin-111 is α1β1γ1 [
]. Previous studies have shown that human liver
express laminins [
] and rat hepatocytes cultured on laminins maintain expression of
hepatic differentiation markers, such as tyrosine aminotransferase, tryptophan-2,
3-dioxygenase, and cytochrome P450 . Identification of the laminin isoforms maintaining viability
and specific functions of human hepatocytes would represent a further advancement.
In the current study, we determined the expressions of laminin chains in isolated primary
human hepatocytes and liver tissue, and investigated the efficacy of human recombinant
laminins on hepatocyte functions in vitro, such as albumin production, hepatic transport activity,
and hepatic metabolism.
Materials and Methods
Isolation and culture of human hepatocytes
Liver tissue was obtained from five patients undergoing liver resection, liver transplantation, or
from deceased organ donors (one female, four males). Donor age ranged from 14 to 74 years
(Table 1). Informed written consent was obtained from all patients or from next of kin on
behalf of children and documented in the patient medical file. The Regional Ethics Committee,
Stockholm, Sweden, approved the study including the consent procedure (Dnr: 2010/678-31/
3). Primary human liver tissue from deceased liver donors were included in the study according
to the regulations of the organ transplantation law of Sweden (1995:831), that is, the donor’s
prior written declaration was followed as well as the written informed consent. Hepatocytes
were isolated using a standardized two-step collagenase perfusion technique as previously
]. Briefly, silicone catheters were sutured into major hepatic vessels on the cut
surface of the tissue. Catheters were connected to a peristaltic pump and the liver specimen
placed in a sterile bag in a water bath at 37°C. The liver specimen was perfused at 10–200 ml/
min depending on the size of the tissue. The first perfusion solution consisted of Hank’s
Balanced Salt Solution (HBSS; without calcium, magnesium and phenol red; Gibco, Life
Technologies, Inc., Rockville, MD, USA) supplemented with 0.5 mmol/L ethylene glycol tetraacetic acid
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Hepatocytes were isolated from 5 different individuals (one female, four males). Liver tissue was obtained from patients undergoing liver resection, liver
transplantation, or from deceased organ donors. Donor age ranged from 14 to 74 years.
(EGTA). After a washout with EGTA free HBSS, the tissue was perfused for 20–30 min with a
third solution composed of Eagle's Minimum Essential Medium (Lonza, Basel, Switzerland)
supplemented with 250 mg/L collagenase Type XI (Sigma-Aldrich, St. Louis, MO, USA) and 50
mg/L DNase (Sigma-Aldrich). Tissue was perfused with solution three for 20–30 min while the
solution was recirculated. Digested tissue was then mechanically disrupted in ice cold Eagle's
minimal essential medium (EMEM). Hepatocytes were pelleted by centrifugation at 70g for 5
min at 4°C. The final hepatocyte pellet was re-suspended in Williams E medium
(SigmaAldrich). Cell number and viability was assessed by standard Trypan blue (Sigma-Aldrich)
Human recombinant laminin isoforms -111, -211, -221, -332, -441, -421, -511, and -521
(BioLamina AB, Stockholm, Sweden), matrigel extracted from Engelbreth-Holm-Swarm murine
sarcoma (EHS; Corning), and collagen type IV (Collagen; Sigma-Aldrich) were pre-coated
on 6, 12, 24, and 96-well plates (BD Biosciences, Mountain View, CA, USA) at least 120 min
Isolated primary human hepatocytes were seeded on plates pre-coated with laminin (10 μg/
ml), EHS, or Collagen (0.3 mg/ml) at a density of 7.5 x 105 cells/ml in Williams E medium
(Sigma-Aldrich) solution supplemented with 100 nM dexamethasone, 2 mM Glutamine
(Sigma-Aldrich), 12 nM insulin (Novo Nordisk Pharma, Malmö, Sweden), 20 mM
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 0.01 M Gentamicin, and 50 nM
Amphotericin B (all from Lonza) at 37°C, with 5% CO2 and in a humidified atmosphere. The laminin
concentration, 10 μg/ml, was selected according to the manufacturer's instructions. The
medium was collected and replaced during the experiments every 24 hours thereafter.
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazolium (MTT;
SigmaAldrich) solution was added to cells in culture medium at a final concentration of 0.5 mg/ml,
and incubated for 30 min. At the end of the incubation period, the medium was removed, and
the converted dye was solubilized with isopropanol. Absorbance of the converted dye was
measured at a wavelength of 590 nm with background subtraction at 650 nm by using Biotek
FLx800™ Multi-Detection Microplate Reader operated by Gen5™ Data Analysis Software.
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Double stranded DNA quantification
Quant-iT™ PicoGreen1 double stranded (ds) DNA kit was used according to manufacturer’s
instructions (Molecular probes, Invitrogen, Camarillo, CA, USA) to quantify dsDNA.
QuantiT™ PicoGreen1 in TE buffer was added to 96-well plate and the fluorescence intensity was
read on a fluorescent spectrometer (Biotek FLx800™ Multi-Detection Microplate Reader) at an
excitation wavelength of 485 nm and an emission wave length of 520 nm. A fresh standard
curve of lambda DNA was analyzed for each assay and results are expressed as mg/ml.
Measurement of human albumin and α1-antitrypsin secretion
Conditioned medium was collected at day six after seeding, and stored at -80°C until analysis.
Human albumin and human alpha-1-antitrypsin (A1AT) were measured by ELISA kit
(Abcam, Cambridge, UK) following protocols supplied by the manufactures. Samples were
collected from each culture condition and each sample was measured in triplicate.
Measurement of bile acids in cell culture medium
Primary bile acids, cholic acid (CA) and chenodeoxycholic acid (CDCA), were analyzed in cell
culture medium as previously described [
]. Briefly, an internal standard composed of
deuterium-labeled cholic acid and chenodeoxycholic acid were added to 1ml of culture medium
collected at day six after seeding. The medium and internal standard, was diluted with 2 mL of
aqueous ethanol (1/1, vol/vol) and hydrolyzed at 120°C for 12 hours using potassium
hydroxide (1 mol/L). The hydrolyzed mixture was diluted with saline and extracted by basic ether
extraction followed by acidic ether extraction. The ether phase was washed with water until
neutral. The solvent was evaporated and the residue was methylated with diazomethane. The
methylated extract was converted into trimethylsilyl ether derivatives with hexamethyl-silazane
and trichlorosilane. Bile acids were measured by gas chromatography/mass spectrometry.
Western blot analysis
Laminin protein levels in isolated hepatocytes and liver tissue were evaluated by western blot
analysis. Isolated hepatocyte and corresponding liver tissue from 6 individuals (Table 2) were
homogenized and sonicated in cracking buffer. Forty μg total protein were mixed with
Laemmli buffer (to a final loading concentration of 2% SDS, 10% glycerol, 0.002%
bromophenol blue, 0.0625 M TrisHCl), supplemented with DTT to a final concentration of 50 mM, and
incubated in 95°C for 5 min. Protein samples and HiMarkTM Pre-stained Protein Standard
(Thermo Fisher Scientific cat # LC5699) were loaded onto Criterion TGX Precast Gels,
4–20% polyacrylamide (Bio-Rad, Hercules, CA) and run at 200 volts on ice for 2 hours. The
proteins were transferred from the gels to PVDF membranes through wet transfer according to
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manufacturer’s protocol (Bio-Rad). To prevent non-specific background binding of the
primary and/or secondary antibodies to the membrane, membranes were blocked in milk-based
blocking buffer (5% (w/v) non-fat dried milk in TBS with 0.1% (v/v) Tween20) for 1 hour. The
primary antibody was diluted (1:1000) in blocking buffer and incubated with the blocked
membranes over-night shaking in 4 degrees Celsius. To remove residual primary antibody, the
membranes were washed 3 x 10 minutes in TBST (TBS with 0.1% (v/v) Tween20). The
secondary antibody for laminin antibodies, HRP-conjugated Goat Anti-Mouse Immunoglobulins
(DAKO cat #P0477), was diluted 1:3000 in blocking buffer and for B-aktin, Amersham ECL
Rabbit IgG HRP-linked F(ab’)2 fragment from donkey (GE healthcare cat # NA9340V), diluted
1:50000 and incubated with the membranes for 1 hour. To remove residual secondary
antibody, the membranes were washed 3 x 10 minutes in TBST. The membranes were incubated
with detection reagent (Super Signal™ West Dura Extended Duration Substrate, Thermo Fisher
Scientific cat # 34075) for 5 minutes. The image was captured using a CCD camera.
Expressions of each laminin isoform were normalized to the value of β-actin expression.
Real time PCR quantification
Total mRNA was isolated from the cultured cells or liver tissue with Trizol (Ambion Life
technologies, Waltham, MA, USA) using the manufactures protocol and dissolved in 50 μl RNase
free water. cDNA synthesis from 1μg RNA was performed by using Applied Biosystem’s High
capacity cDNA reverse transcription kit. Quantification of mRNA was performed using
TaqMan real time PCR, employing the Applied Biosystems Step one plus Real-Time PCR system.
Relative mRNA expression was calculated from the Ct-values against the house keeping gene
PPIA using the comparative delta-Ct method. The following primers targeting specific mRNA
were used: Hs99999904_m1 (peptidyl-prolyl cis-trans isomerase A; PPIA), Hs00910225_m1
(albumin; Alb), Hs01097800_m1 (alpha-1-antitrypsin; A1AT), Hs00230853_m1 (hepatocyte
nuclear factor 4 alpha; HNF4a), Hs00609178_m1 (glucose-6-phosphate; G6P), Hs00430021_
m1 (cytochrome P450 3A4; CYP3A4), Mm00484150_m1 (cytochrome P450 7A1; CYP7A1),
Mm01278617_m1 (Ki67), and Hs01385685_m1 (multidrug resistance-associated protein 2;
Normal distribution was assessed by skewness (histograms). Due to a low number of samples
and skewed data non-parametric Kruskal-Wallis with Dunn’s multiple comparison test to
correct for multiple comparisons was used to evaluate differences between samples. A p-value
of < 0.05 was considered statistically significant and all calculations were performed using
GraphPad Prism1 software (GraphPad Software Inc., San Diego, CA).
Expression of laminin in liver tissue and isolated hepatocytes
The protein expressions of the different laminin chains in human liver and isolated hepatocytes
were evaluated by western blot analysis. Laminin chains of α2, α3, α4, β1, β3, γ1, and γ2 were
detected both in liver tissue and isolated hepatocytes, and α1 and α5 were absent in both.
Laminin chain of β2 was tested with two different antibodies but was inconclusive due to multiple
bands. The expressions of laminin were stronger in liver tissue than in isolated hepatocytes,
and there were individual variations between patients (Fig 1).
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Fig 1. Expression of laminin in human liver and isolated hepatocyte. Expression of laminin was determined in protein samples from
liver tissue (LT) and isolated hepatocytes (HC) by western blot analysis. (A; Laminin-α1, B; Laminin-α2, C; Laminin-α3, D; Laminin-α4, E;
Laminin-α5, F; Laminin-β1, G; Laminin-β2, H; Laminin-β3, I; Laminin-γ1, J; Laminin-γ2, K; β-actin) Phenotypes of laminins, α2, α3, α4, β1,
β3, γ1, and γ2 were detected both in liver tissue and isolated hepatocytes, and α1 and α5 were absent in both. Laminin chain of β2 was
inconclusive due to multiple bands. Sample number (#1–#6) is corresponding to Table 2. Values were normalized to the level of β-actin in
Isolated primary human hepatocytes were cultured on different laminin isoforms -111, -211,
-221, -332, -411, -421, -511, and -521, EHS, or Collagen pre-coated plates, and were evaluated
morphologically. Hepatocytes cultured on EHS migrated within a few days and formed
threedimensional cell clusters while hepatocytes cultured on laminin or Collagen showed
characteristic, hexagonal shape in a flat cell monolayer for up to six days (Fig 2). Morphological
differences in shape, density, and appearance of nuclei between different laminin isoforms were
seen, however, this was difficult to measure.
Viability of hepatocytes
Hepatocyte viability was assessed by MTT assay at day six after seeding. All laminin isoforms
maintained hepatocyte viability comparable to Collagen and EHS. No statistical differences
between the different laminins, EHS and Collagen were seen (Fig 3).
dsDNA concentrations and mRNA expression of Ki67
The dsDNA concentrations (Fig 4A) and mRNA expression of Ki67 (Fig 4B) were evaluated in
cultured hepatocytes at day six after seeding. No significant difference in dsDNA concentration
or Ki67 expression was seen between the different laminin isoforms and the values were
comparable to the values seen for hepatocytes cultured on EHS or Collagen.
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Fig 2. Morphology of primary human hepatocytes seeded on laminin, EHS, or Collagen. Isolated
primary human hepatocytes were seeded on laminin isoforms -111, -211, -221, -332, -411, -421, -511, and
-521, EHS, or Collagen pre-coated plates. Hepatocytes cultured on EHS coated plates migrated and formed
three-dimensional structures. Hepatocytes cultured on laminins and collagen showed a hexagonal shape in a
flat cell monolayer. Isolated human hepatocytes could be incubated on laminins for up to six days without
obvious morphological changes.
Production of human albumin and A1AT
We evaluated liver functions by human albumin and human A1AT synthesis and mRNA
expression. As shown in Fig 5, hepatocytes cultured on laminin maintained mRNA expression
and protein secretion of albumin and A1AT equally well compared to those of hepatocytes on
EHS or Collagen.
Fig 3. Viability of cultured hepatocytes. Hepatocyte viability during the culture was assessed at day six
after seeding by MTT assay. Each laminin isoform supports hepatocyte viability during culture for at least 6
days, and no statistical differences between each laminin, EHS and Collagen were seen. The results are from
five replicates and are expressed as mean ± SEM.
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Fig 4. dsDNA concentrations and mRNA expression of Ki67. The dsDNA concentrations (A) and mRNA
expression of Ki67 (B) were evaluated at day six after seeding. No significant differences in dsDNA or Ki67
expression could be noted between different laminin isoforms, Collagen and EHS. The results from five
replicates are expressed as mean ± SEM.
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Fig 5. Productions and mRNA expressions of human albumin and A1AT. Production and mRNA
expression of human albumin (Fig 5A) and human alpha-1-antitrypsin (A1AT; Fig 5B) were evaluated by
ELISA and RT-PCR at six days after seeding. Hepatocytes cultured on laminin maintained the production
and mRNA expressions of albumin and A1AT as compared to those of hepatocytes on EHS or Collagen. No
significant differences in the productions and mRNA expressions of human albumin and human A1AT could
be noted among different laminin isoforms, Collagen and EHS. The results from five replicates are expressed
as mean ± SEM.
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Bile acid synthesis
Bile acid synthesis was assessed as a liver-specific function of primary human hepatocytes.
Hepatocytes cultured on laminin showed maintained bile acid production as measured by
cholic acid (CA) and chenodeoxycholic acid (CDCA) in medium at day six after seeding. The ratio
of CDCA and CA was similar in all groups. There was no significant difference in the
production of CDCA or CA among different laminin isoforms, Collagen and EHS (Fig 6A). To assess
the bile acid synthesis, mRNA expression of CYP7A1 was also evaluated; CYP7A1 is the
ratelimiting enzyme in the first step of classical pathway in bile acid synthesis [
]. The mRNA
expression of CYP7A1 showed a similar trend of the total amount of CA and CDCA
synthesized; where hepatocytes cultured on EHS, laminin-111, -332, and -411 showed higher mRNA
expression of CYP7A1 (Fig 6B).
Expression of metabolic genes in isolated hepatocyte
To evaluate the expression of genes involved in metabolic functions of hepatocytes, mRNA
expression of CYP3A4, HNF4a, G6P, and MRP2 of hepatocytes were assessed. Hepatocytes
cultured on laminins demonstrated no significant differences in mRNA expressions of
CYP3A4, HNF4a, G6P, or MRP2 as compared to those of Collagen or EHS (Fig 7A–7D).
Defined culture system, free of animal components is of great importance for the development
of hepatocyte research and regenerative therapy. Conventionally, hepatocytes are often
cultured on non-defined matrices, such as EHS (trade name Matrigel™). The Matrigel™ matrix is
of animal origin and consist of a mix of several basement membrane proteins, such as
laminin111, collagen IV, nidogen/enactin and proteoglycan [
] which makes it unsuitable for
controlled and defined culture systems. In this study, we evaluated the efficacy of human
recombinant laminins as a defined and xeno-free alternative, culture matrix for primary human
hepatocytes. We demonstrated that all laminin isoforms tested in this study, -111, -211, -221,
-332, -411, -421, -511, and -521, retained viability, gene expressions, and functional properties
of primary human hepatocytes for up to 6 days in culture, and of note, these results are
comparable to hepatocytes cultured on conventionally used animal derived EHS or Collagen. To our
knowledge, this is the first report of culturing and characterizing primary human hepatocytes
on human recombinant laminins. Hepatocytes cultured on laminin and Collagen showed
characteristic, hexagonal shape in a flat cell monolayer while hepatocytes cultured on EHS rapidly
migrated and formed three-dimensional cell cluster. The three-dimensional structure makes it
hard to compare the EHS results to the other matrices as three-dimensional culture induces
many metabolic functions. An important functional test was to determine whether primary
human hepatocytes would maintain the ability to produce bile acids. As shown, hepatocytes
cultured on laminin produced cholic acid and chenodeoxycholic acid. The mRNA expression
of CYP7A1, the rate-limiting enzyme of the classical pathway in bile acid synthesis, and
HNF4a, which regulates CYP7A1 mRNA expression [
], remained unaltered during the
culture periods when compared to those of hepatocytes cultured on EHS or Collagen.
Laminins are large trimeric proteins that contain α-chain, β-chain and γ-chain, that can be
found in five, three and three genetic variants, respectively. In the human body, at least 16
laminin isoforms with distinct specific roles in cell maintenance adhesion, cell differentiation and
migration have been identified [
]. The distribution of laminin chains in human liver tissues
is still not fully evaluated but it has been demonstrated that laminin α5 chain is widely
observed, except in the sinusoids. The other α chains are variously expressed in Glisson's
sheath and central veins. Laminin chains β1, β2, and γ1 chains were also widely observed [
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Fig 6. Bile acid synthesis. Bile acid (CA and CDCA) concentrations in the medium were assessed at six
days after seeding. All of hepatocytes seeded on laminins, EHS, or Collagen demonstrated almost same ratio
of CDCA and CA. In terms of the amount of CA, hepatocytes cultured on EHS produced more compared to
cells cultured on laminin-211, -411, -421, -511, and -521, however there was no significant difference in the
concentration of CDCA or CA among different laminin isoforms, Collagen and EHS. (A). The mRNA
expression of CYP7A1 showed a similar trend of the total amount of CA and CDCA synthesized; hepatocytes
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cultured on EHS, laminin-111, -332, and -411 showed higher mRNA expression of CYP7A1 (B). There was
no significant difference in the mRNA expression of CYP7A1 among different laminin isoforms, Collagen and
EHS. The results are from five replicates and are expressed as mean ± SEM.
In the current study, we evaluated the expression levels of laminin, and found that protein of
laminin α2, α3, α4, β1, β3, γ1, and γ2 were detected both in liver tissue and isolated
hepatocytes, and α1 and α5 were absent (Fig 1). The discrepancy between the previous study and our
current results may be explained by differences in detection methods; immune histochemical
analysis or western blot analysis. The intra-individual variability seen in the 6 samples
examined in this study also suggests that the discrepancy could be due to variability in patient
material and underlying liver diseases that may affect the expression of laminins. Studies on human
laminin diseases, such as Pierson syndrome [
] and congenital muscular dystrophy type 1A
], or gene inactivation in animals [
] have provided knowledge about the detail properties
and biological roles of each isoforms of laminin. However, the detailed functions of each
laminin-isoforms associated with the liver tissue or isolated hepatocytes remain unclear. Due to
coexistence and highly cross-linkage between laminin isoforms [
], partial compensation by
others isoforms may mask the specific function. Moreover, different laminin isoforms have
common cell adhesion properties, like common integrin binding sites (15). Indeed, in this
study, despite no protein expressions of α1, and α5, hepatocytes cultured on isoform of
laminin-111, -511, and -521 maintained hepatic functions, such as production of albumin, A1AT,
and bile acids and, mRNA expression of CYP3A4, CYP7A1, and G6P.
Laminins also play important roles in many different cellular processes, and
laminininduced cell signaling can influence on gene transcriptions and re-modeling of promoter
chromatin leading to proliferation, differentiation and maintenance of different tissues [
Pluripotent stem cells derived from the inner cells mass also express laminin chains, and
pluripotency of human pluripotent stem cells can be long-term maintained in vitro on laminin-521
or -511 without loss of pluripotency and with maintained karyotype [
]. Likewise, it has
been increasing recognized that different laminin isoforms are critically important for
maintenance and development of different tissues, for example; epithelial cells need laminin-332
together with laminin-511/521, muscle and nerve cells require laminin-211, -221 and -511/521,
and endothelial cells grow on laminin-411 in combination with laminin-511 .
Hepatoblastlike cells could be maintained long-term when cultured on laminin-111 with the ability to
differentiate into both hepatocyte-like cells and cholangiocyte-like cells [
]. The understandings
of functional properties of each isoforms of laminin on primary human hepatocytes would
provide manageable tools for cell sources of hepatic regenerative therapy, and for hepatocytes
From hepatocyte transplants point of view, it has demonstrated that human hepatocytes
graft transplanted into the mouse subcutaneous space or under the kidney capsule survived
significantly longer-period when extracellular matrix components were provided to the grafts
]. It has been demonstrated that elevated CYP3A4 activity in hepatocytes grown on matrices
or encapsulated to create a 3-dimentional environment [
]. The mRNA expression of
CYP3A4 in hepatocytes cultured on laminin-111 and -332 in our study could be associated
with favorable cell-matrix integrin binding, increased cell-to-cell contact additional secreted
extracellular matrix and/or recovery of cell polarity [
]. Given the notion that Matrigel™ is
rich in laminin-111, and that it is effective for cell attachment and differentiation of
hepatocytes in vitro, hepatocyte transplantation together with human recombinant laminin would be
a promising xeno-free strategy for improvement for the outcome of clinical hepatocyte
transplantation. Such studies are currently under progress.
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Fig 7. Metabolic activity of isolated hepatocyte. The mRNA expression of CYP3A4 (A), HNF4a (B), G6P
(C), and MRP2 (D) of isolated hepatocytes cultured on laminin were assessed. Hepatocytes cultured on
laminins demonstrated no significant differences in mRNA expressions of CYP3A4, HNF4a, G6P, or MRP2
as compared to those of Collagen or EHS. The results are from five replicates and are expressed as
mean ± SEM.
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In summary, primary human hepatocytes cultured on human recombinant laminins showed
comparable liver-specific functions compared to those of EHS or collagen. Recombinant
laminins offer a xeno-free alternative of long-term culture of primary human hepatocytes allowing
its use in hepatocyte regenerative medicine.
S1 File. Data set of Figs 1, 3, 4, 5, 6 and 7.
Conceived and designed the experiments: MW EE.
Performed the experiments: MW HJ HZ CJ ML.
Analyzed the data: MW HZ HJ EE.
Contributed reagents/materials/analysis tools: LH HZ.
Wrote the paper: MW.
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