Liver tissue fragments obtained from males are the most promising source of human hepatocytes for cell-based therapies – Flow cytometric analysis of albumin expression
Liver tissue fragments obtained from males are the most promising source of human hepatocytes for cell-based therapies ± Flow cytometric analysis of albumin expression
Karolina Ewa Zakrzewska 0 1 2 3
Anna Samluk 0 1 2 3
Agnieszka Wencel 0 1 2 3
Krzysztof Dudek 0 2 3
Dorota Genowefa Pijanowska 0 1 2 3
Krzysztof Dariusz Pluta 0 1 2 3
0 Funding: Nalecz Institute of Biocybernetics and Biomedical Engineering, Polish Academy of Sciences statutory funding
1 Department of Hybrid Microbiosystems Engineering, Nalecz Institute of Biocybernetics and Biomedical Engineering, Polish Academy of Sciences , Warsaw , Poland , 2 Chair and Department of General, Transplant and Liver Surgery, Medical University of Warsaw , Warsaw , Poland
2 Editor: Ezio Laconi, Universita degli Studi Di Cagliari , ITALY
3 Source Bethyl Laboratories, Inc. , Montgomery, TX, USA (A80-129F) Bethyl Laboratories , Inc. , Montgomery, TX, USA (A80-122F) Becton Dickinson, Warsaw, Poland (345809) Becton Dickinson, Warsaw, Poland (345787) Abcam, Cambridge, UK (ab37374) Abcam, Cambridge, UK, ab46739
Cell-based therapies that could provide an alternative treatment for the end-stage liver disease require an adequate source of functional hepatocytes. There is little scientific evidence for the influence of patient's age, sex, and chemotherapy on the cell isolation efficiency and metabolic activity of the harvested hepatocytes. The purpose of this study was to investigate whether hepatocytes derived from different sources display differential viability and biosynthetic capacity. Liver cells were isolated from 41 different human tissue specimens. Hepatocytes were labeled using specific antibodies and analyzed using flow cytometry. Multiparametric analysis of the acquired data revealed statistically significant differences between some studied groups of patients. Generally, populations of cells isolated from the male specimens had greater percentage of biosynthetically active hepatocytes than those from the female ones regardless of age and previous chemotherapy of the patient. Based on the albumin staining (and partially on the α-1-antitrypsin labeling) after donor liver exclusion (6 out of 41 samples), our results indicated that: 1. samples obtained from males gave a greater percentage of active hepatocytes than those from females (p = 0.034), and 2. specimens from the males after chemotherapy greater than those from the treated females (p = 0.032).
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
Competing interests: The authors have declared
that no competing interests exist.
According to data available from the Centers for Disease Control and Prevention chronic liver
disease and cirrhosis are the 12th leading causes of death in the United States (1999±2014) [
Liver transplantation is a viable treatment option for end-stage liver disease and acute liver
failure. However, a shortage of donors reduces the advantages of this procedure. Every year, in
the United States, of the 15 000 people waiting for a liver transplant, 10% die [
such as hepatocyte transplantation and use of bioartificial liver devices could represent feasible
therapies bridging the time for whole organ transplantation or supporting the regeneration of
the liver [
]. All these alternatives require an adequate source of functional hepatocytes.
Successful isolation of primary human hepatocytes remains a challenge and comparative
studies need to be carried out in cells derived from different sources. The majority of published
studies have focused on two aspects of the problem: cell yields and viability. Physiological
activity or synthetic capacity of the isolated hepatocytes still pose a poorly recognized problem.
It is commonly accepted that warm and cold ischemia time are one of the most important
factors affecting hepatocytes isolation [4±7]. Type of the liver tissue (resected fragments, donor
liver, diseased liver, cirrhotic liver, etc.) as well as preoperative blood tests and histopathology
can also be a predictive factor for determining the likelihood of a successful isolation [6±11].
On the other hand, there is little scientific evidence for the influence of patient's age, sex, and
chemotherapy on the quality of the harvested hepatocytes. Moreover, published research
results are conflicting [7,10±13].
In this paper, we present a comparative study on the impact of the age, sex, and
chemotherapy on the yield and viability of the metabolically active hepatocytes. We used a method based
on immunostaining of cells and flow cytometry. To our best knowledge, the method developed
to estimate quality, in particular ability to produce albumin and α-1-antitrypsin, of the isolated
hepatocytes by the flow cytometric analysis is published for the first time. Our results enabled
us to rank sources of hepatocytes by the synthetic capacity of the isolated cells.
Patients and methods
Liver tissue was obtained from surgical procedures carried out at the Department of General,
Transplant and Liver Surgery, Medical University of Warsaw, Poland (October 2012 to March
2016). Ethical approval for the study was granted by the Local Research Ethics Committee
(reference number KB/182/2008). Study was performed according to the principles of the 1975
Declaration of Helsinki. None of the transplant donors were from a vulnerable population and
all donors or next of kin provided written informed consent that was freely given.
Human liver cells isolation
Human liver cells were isolated from fragments of normal tissue obtained from hepatic
resections carried out for metastatic disease (colon/ovaries/breasts), primary liver cancer or other
reasons (usually benign tumors or another type of liver disease). Some of the isolations were
performed using tissue fragments from livers not suitable for transplantation (D, donor liver).
Twenty six of 41 patients enrolled in the study had received perioperative chemotherapy
(13 women and 13 men). Five different treatment regimens had been applied: FOLFOX4 (3
women and 3 men), FOLFOX6 (3 women and 2 men), FOLFIRI (2 women and 6 men), LF4 (3
women and 1 man), and XELODA (2 women and 1 man).
In 10 patients who had received chemotherapy (10/26) 6 of their livers showed signs of
steatohepatitis, 2Ðsigns of fibrosis, and 2Ðsigns of steatosis. Additionally, two individuals (2/41)
had diabetes. None of the patients had cirrhosis.
The type of liver tissue and the results of cells isolations are presented in Table 1.
The isolation of liver cells was carried out using a method based on enzymatic tissue
disaggregation. Following resection, the fragments were immediately placed in a sterile sealed string
bag containing ice-cold University of Wisconsin (UW±ViaSpan; DuPont Industrial
Biosciences, Wilmington, DE, USA) solution and processed within 1 hour. In cases of donor liver
fragments, the specimens have been kept in sterile UW solution on ice for several hours (less
than 12 hours). The isolation started with the washing of liver fragments through the exposed
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* colon/ovaries/breastsÐmetastasis of colon/ovaries/breasts cancer to the liver; donor±liver not used for transplant; liver±primary liver cancer; other±cases
which do not fit into mentioned above
** median values with the range (in parentheses)
3 / 14
vessels with ice-cold phosphate-buffered saline (PBS, without Ca2+ and Mg2+, pH 7.2; Lab
Empire, Rzeszow, Poland) until all remaining blood was removed (flow rate 10 mL/min).
Next, the tissue was minced with a scalpel in pre-warmed (37ÊC) 0.05% collagenase I (Life
Technologies, Warsaw, Poland) solution in the William's E Medium (Sigma-Aldrich, Poznan,
Poland) and kept under constant agitation on a magnetic stirrer at 37ÊC for 1 hour. The
obtained cell suspension was filtered through the cell strainer with 70 μm nylon mesh
(Corning Inc., Riverfront Plaza, NY, USA). After centrifugation at 366xg (7 min, 4ÊC), the residual
erythrocytes were destroyed by the incubation of the pellet in the red blood cell lysis buffer
solution (155mM NH4Cl, 10mM NaHCO3, and 0.1mM EDTA; POCH, Gliwice, Poland) at
37ÊC for 5 min. Next, the cells suspension was centrifuged over a Percoll (GE Healthcare
BioSciences, Uppsala, Sweden) cushion (1.04 g/ml) at 366xg (20 min, 4ÊC) to remove cell debris.
Finally, after a washing step, the cell pellet was resuspended in the Dulbecco's Modified Eagle
Medium (Sigma-Aldrich, Poznan, Poland) supplemented with 10% fetal bovine serum (FBS;
Biological Industries, Kibbutz Beit-Haemek, Israel). Viable and dead cells were discriminated
and counted using the 0.4% Trypan blue (Sigma-Aldrich, Poznan, Poland) staining with a
Populations of the cells isolated from liver tissue were analyzed using flow cytometry (FACS,
fluorescence-activated cell sorting). The immunostaining of the cells was described elsewhere
]. Briefly, cells were fixed with 4% formaldehyde and permeabilized using 0.1% Triton
X100 solution in PBS. Prepared cells (1 × 106 per sample) were incubated with
fluorophore-conjugated specific antibodies (Table 2) for 1 hour at room temperature. Samples were analyzed
immediately after staining with FACSCanto II flow cytometer (Becton Dickinson, Warsaw,
Poland). Single immunostainings were used for the assessment of hepatocytes' biosynthetic
capacity. Unstained specimens and cells incubated with appropriate isotype immunoglobulins
(Table 2) were used as negative controls.
Human hepatoma C3A (ATCC No. CRL-10741) and osteosarcoma HOS (ATCC No.
CRL1543) cell lines were used to verify the specificity of antibodies against hepatic markers.
FACSDiva software (Becton Dickinson, Warsaw, Poland) was used for data acquiring and analysis.
Patients and statistical analysis
Thirty five patients from whom the liver tissue samples were collected (Table 1; donor livers,
n = 6, excluded from the statistical analysis) were divided into the following groups: males (M)
of size of 18 patients, females (F) = 17, without chemotherapy (Ch-) = 9, with chemotherapy
(Ch+) = 26, under 60 years old (A60-) = 18, 60 years old and older (A60+) = 17, and certain
combinations of above-mentioned ones. The group of 4 patients was the smallest one used for
comparison (F Ch-).
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Data were analyzed using R version 3.2.2. R is a Free Software under the terms of the
GNU General Public License [
]. The differences between two independent groups were
analyzed using the Welch's two-sample t-test. For multiple comparisons the Benjamini &
Hochberg correction procedure [
] was applied. In multivariate analysis the two-way
Analysis of Variance (ANOVA) for linear model with interaction term was used. ANOVA
results were subsequently confirmed using the Tukey HSD test [
]. The p-values lower
than 0.05 were considered as statistically significant. The results are presented as the median
values with the range for each variable.
Populations of cells isolated from liver tissue
The rapid and low-stress method for the isolation of all cells from liver tissue gave high yields
(3.1 × 106/g (1.1±12.0)) of viable cells (97.2% (91.8%±99.9%)) (Table 1). FACS analysis of the
isolates using forward and side light scatter revealed the presence of minimum two and
maximum four distinct cells populations: P2 ±P5, depending on specimen (Fig 1).
The hepatocytes are present in the P2. Nonparenchymal cells (liver sinusoidal endothelial
cells, Kupffer cells, lymphocytes, biliary cells, stellate cells, various progenitors, etc.) belong to
the other populations. The identity of the hepatocytes was confirmed by labeling with
fluorophore-conjugated albumin (Alb)- and α-1-antitrypsin (A1AT)-specific antibodies (Fig 2A).
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Fig 2. Staining of the isolated cells with fluorophore-conjugated specific antibodies. Exemplary
immunostainings of the cells isolated from liver specimen (H22-13, see Table 1). (A) The cells labeled with
anti-Alb (left panel) and anti-A1AT (right panel) antibodies were found predominantly in the P2 population
(97.5% and 86.2% of the P2, respectively) and identified as hepatocytes. (B) Nonparenchymal liver cells were
stained with anti-CD45 (populations P3 = 80.3% and P4 = 74.1%, left panel) and anti-CD14 antibodies
(population P4 = 43.4%, right panel) (see Table 2 for the list of antibodies). Alb- and A1AT-positive cells±
green dots; CD45-positive cells±pink dots; CD14-positive cells±blue dots. Abbr.: Alb, albumin; A1AT,
α1-antitrypsin; FSC, forward scatter; SSC, side scatter.
The populations of cells other than P2 were labeled with anti-CD45 and anti-CD14 antibodies
This staining is not sufficient to discriminate between different cell types populating liver
but eventually allows for distinguishing parenchymal from nonparenchymal cells. As shown in
Fig 3A, Alb-positive cells were labeled neither with anti-CD45 nor with anti-CD14 antibodies.
On the other hand, some fractions of nonparenchymal cells exhibited immunoreactivity for
both CD markers (Fig 3B). The CD45 and CD14 can be common markers for the Kupffer cells
Differential percentage of cells in P2 population
As expected, after the initial removal of most of the blood cells from the tissue fragment,
hepatocytes represent a large fraction of the isolated cells. A median percentage of cells belonging
to the P2 population in all samples (n = 41) was 47.6. There were differences between groups
observed in the entire patients' population (see Patients and Methods section for description
of cohorts), yet the statistically significant difference (p = 0.002) was observed only between
donors (n = 6) and non-donors (n = 35) groups (64.1% (55.2%±86.2%) and 38.5% (14.6%±
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Fig 3. Double immunostaining of the isolated cells. Exemplary double immunostainings of the cells
isolated from liver specimen (H31-14, see Table 1). (A) In the P2 population cells labeled with anti-Alb
antibody expressed neither CD45 (83.5% vs.1.3%, left panel) nor CD14 (81.3% vs. 0.5%, right panel)
markers. (B) Nonparenchymal cells in the P3 population (left panel) expressed CD45 marker but not CD14
(46.7% vs. 0.1%). Some of the cells from the P4 population (right panel) expressed both CD45 and CD14
markers (CD45 = 23.9%, CD14 = 16%, and CD45/CD14 = 15.1%) (see Table 2 for the list of antibodies).
Albpositive cells±green dots; CD45-positive cells±pink dots; CD14-positive cells±blue dots; CD54/CD14-positive
cells±grey dots; unstained cells±yellow dots. Abbr.: Alb, albumin; FITC, PerCP, APCÐfluorophores.
81.2%), respectively). This above-average percentage of the P2 cells in the isolates from the
donor livers is probably resulting from the different procedure applied for the pre-preparation
of the whole donor organs. Nevertheless, the donor group was omitted from further statistical
analysis, because it is not representative of the entire patients' population (males only) and we
found this tissue not suitable for hepatocytes' isolation (this issue is discussed in the following
Generally, samples obtained from females (F = 17) gave greater percentage of the cells
belonging to the P2 population then those from males (M = 18) (47.6 (22.0±81.2) and 32.9
(14.6±75.3), respectively; p = 0.162), from people with no chemotherapy (Ch- = 9) greater
than from treated (Ch+ = 26) patients (40.6 (18.8±75.3) and 35.1 (14.6±81.2), respectively;
p = 0.823), and from younger people (60- = 18) greater than from older ones (60+ = 17) (48.6
(18.7±81.2) and 32.3 (14.6±75.2), respectively; p = 0.186). The two-way ANOVA model
detected no interactions between above variables. The broad range of the percentage of cells
found in the P2 population (14.6±81.2) is notable and suggests that these outcomes can be
affected by both the quality of the tissue and by the effectiveness of blood removal (variable
size of the P3±P5 populations). Thus, the meaning of these results is not clear and is not further
7 / 14
Production of albumin and α-1-antitrypsin by the hepatocytes
Since the production of Alb and A1AT is a commonly accepted measure of metabolic capacity
and viability of hepatocytes we have focused on these two markers. When all tissue samples
were taken into account (n = 35), a median percentage of Alb-positive cells in the P2
population was 85.8 (56.5±98.7) and A1AT-possitive (data available for 29 samples: H9-13 ±
H4416)± 88.9 (30.4±97.1).
Impact of sex and chemotherapy. In the single variable comparison, the difference in the
percentage of Alb-positive hepatocytes in the P2 population between males (M = 18: 86.9
(59.9±98.7)) and females (F = 17: 80.3 (56.5±97.5)) was statistically significant (p = 0.038) (Fig
4A, left panel). Interestingly, it seems that chemotherapy did not affect the percentage of
Albpositive hepatocytes when the entire studied population was analyzed (Ch- = 9: 84.0 (59.9±
97.5) and Ch+ = 26: 86.1 (56.5±98.7), p = 0.902) (Fig 4A, middle panel). However, when the
groups of males and females with or without chemotherapy were compared, the impact of
chemotherapy was apparent (p = 0.04 after correction) between M Ch+ = 13 (87.3 (82.1±98.7))
and F Ch+ = 13 (80.3 (56.5±97.3)), but not significant (p = 0.61 after correction) between
nontreated patients: M Ch- = 5 (84.0 (59.9±94.5)) and F Ch- = 4 (86.6 (73.4±97.5)) (Fig 4B).
Additionally, it appeared that chemotherapy had no effect in the patients within the same sex
groups (M Ch+/M Ch-: p = 0.40 and F Ch+/F Ch-: p = 0.40).
In order to investigate the exact interactions between these two variables the two-way
ANOVA test was performed. The p-value for the interaction term ªM/Fº was significant
(0.034), whereas for term ªSex/Chemotherapyº it was not (0.058). However, it is close to the
significance level and the interaction plot (S1 Fig) clearly reveals non-parallel lines, which
indicates possible interaction between these variables. The ANOVA results were then confirmed
using the Tukey HSD test. The p-value for the difference between males and females was 0.034
and for M Ch+ and F Ch+ groups± 0.032 (Table 3).
Impact of age and sex. The age of the patients analyzed individually had no effect on the
percentage of Alb-positive cells in the P2 population: A60- = 18 (86.7 (59.9±98.7)) and A60+ =
17 (84.7 (56.5±97.5)), p = 0.547 (Fig 4A, right panel). The difference was visible for M A60- = 7
(92.6 (81.2±98.7)) and M A60+ = 11 (85.8 (59.9±97.5)), yet this difference was not statistically
significant (p = 0.42 after correction). Such a difference was not noticeable for females (F A60- = 11:
80.3 (59.9±97.5) and F A60+ = 6: 79.8 (56.5±95.9), p = 0.52 after correction), but was
considerable (although not statistically significant±p = 0.19 after correction) between M A60- and F A60-.
In the group of A60+ patients (n = 17) the difference between males and females was smaller and
not significant (p = 0.32 after correction) (Fig 4C). In the multivariate analysis, the two-way
ANOVA test again showed significant interaction for ªM/Fº (p = 0.039 after Tukey HSD test)
and no interaction between the sex and the age of the patients (p = 0.772) (Table 3).
Staining of the P2 cells with the A1AT antibody in part confirmed the results obtained for
the Alb-positive cells. However, in the case of the percentage of the A1AT-positive cells, all
three variables: sex, chemotherapy, and age analyzed individually seem to have no impact on
the differences in studied groups. Some differences, even if visible, did not reach statistical
significance: M = 14: 89.4 (41.4±97.1) and F = 15: 83.5 (30.4±95.8), p = 0.79; Ch+ = 22: 84.6
(30.4±97.1) and Ch- = 7: 92.5 (61.7±96.5), p = 0.092; A60- = 16: 89.2 (30.4±97.1) and A60+ =
13: 81.5 (41.4±95.8), p = 0.20 (Fig 5). Consequently, in multivariate analysis no statistically
significant interactions were detected between these variables.
In order to simplify and hasten the procedure for liver cells isolation we have developed the
method based on enzymatic tissue disaggregation. It provided high yields of cells, comparable
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Fig 4. The impact of sex, chemotherapy, and age on the percentage of Alb-positive cells in P2 population.
Comparison of the percentage of Alb-positive cells in the P2 population between groups of patients. (A) F (n = 17), M
(n = 18); Ch- (n = 9), CH+ (n = 26); A60- (n = 18), A60+ (n = 17). (B) F Ch+ (n = 13), M Ch+ (n = 13), F Ch- (n = 4), M
Ch(n = 5). (C) F A60- (n = 11), F A60+ (n = 6), M A60- (n = 7), M A60+ (n = 11). The differences between two independent
groups were analyzed using the Welch's two-sample t-test. For multiple comparisons the Benjamini & Hochberg
correction procedure was applied. In multivariate analysis the two-way Analysis of Variance (ANOVA) for linear model with
interaction term was used. ANOVA results were subsequently confirmed using the Tukey HSD test. The results are
presented as the median values with the range for each variable. * p < 0.05 Abbr.: Alb, albumin; F, female; M, male; Ch-,
non-treated patients; Ch+, patients after chemotherapy; A60-, patients under 60 years old; A60+, patients 60 years old
to those presented in the available literature, with high viability [4±11,18,19]. However, this
technique is rather appropriate for small tissue fragments (5±100 g) and does not include the
step for hepatocytes enrichment. The classical and the most commonly used two-step
collagenase perfusion, described for the first time by Seglen (1976) [
], is more suitable for the
whole-organ or large-scale isolations. In spite of that, the presented method corresponds to the
requirements of the flow cytometric analysis: fast and low-stress sample preparation. Specific
isolation of the hepatocytes is not necessary since the desired cell population can be set apart
using a cytometer.
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M Ch+/F Ch+
We have observed only minor differences in both isolation efficiency and cells viability
between analyzed groups. Neither age of the tissue donors, sex, and chemotherapy nor
combinations of above-mentioned parameters gave p-values equal or lower than 0.05. In the study of
criteria suitable for the identification of the most promising liver specimen Vondran et al.
(2008) isolated human hepatocytes from tissue after partial hepatectomy (n = 50). As it was
shown in our report, the patient's sex and previous chemotherapy had no influence. Contrary
to our results, donor age significantly affected the isolation outcome, but it was correlated with
the overrepresentation of the primary and secondary tumors in the group of older patients.
Thus, the quality of the individual specimen rather than the age was important and this
parameter was not found suitable for predicting cell yields [
]. Also preoperative blood tests and
histopathology (e.g. GGT levels, cholestasis, cirrhosis, and steatosis) should have a predictive
potential, but in these fields results are conflicting [6±11]. Nevertheless, we have not analyzed
the correlation between the indications for surgery and the outcome of isolation since there
were no differences either in the cell yield (p = 0.669) or the viability (p = 0.303) between
specimens from the younger and the older patients. However, in a study including liver pieces from
as many as 1034 individuals, carried out in order to establish an algorithm that predicts the
yield and viability of isolated hepatocytes, statistically significant differences between various
groups have been found. After multiple regression analysis, apart from the tissue quality
correlated with the disease, such variables as chemotherapy, sex, and age influenced the analyzed
parameters. This study detected statistical significance in more variables than any other
research due to the remarkable increase in the statistical power [
]. The authors indicated that
their analysis was able to pick up a statistical significance where other studies found lack of
interactions possibly because of a greater number of replicates. Moreover, they observed a loss
of statistical power when multiple regression analyses were performed on the samples with
reduced sizes (218 for viability and 128 for yield of the isolated cells). Thus, in our case, it is
possible that the smaller number of analyzed tissue fragments was the reason for the lack of
statistical significance in the liver cells' yield and viability between the examined groups of
In our report, median isolation efficiency and cell viability in the group of liver donors
(n = 6) were lower than those obtained in the rest of the population (n = 35): 2.7 × 106/g and
96.7% vs. 3.4 × 106/g and 97.3%, respectively. These results, although not statistically
significant (p = 0.209 and p = 0.19, respectively), are in line with others. Bhogal et al. (2011) in the
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Fig 5. The impact of sex, chemotherapy, and age on the percentage of A1AT-positive cells in P2 population.
Comparison of the percentage of A1AT-positive cells in the P2 population between groups of patients. F (n = 15), M
(n = 14), Ch- (n = 7), Ch+ (n = 22), A60- (n = 16), A60+ (n = 13). The differences between two independent groups were
analyzed using the Welch's two-sample t-test. The results are presented as the median values with the range for each
variable. Abbr.: A1AT, α-1-antitrypsin; F, female; M, male; Ch-, non-treated patients; Ch+, patients after chemotherapy;
A60-, patients under 60 years old; A60+, patients 60 years old and older.
study on isolation of the cells from 104 liver fragments reported lower viability and yield of
hepatocytes from donor livers (n = 7) due to the longer cold ischemia time that donor livers
have been exposed to when compared to normal livers [
]. In the research results presented by
Hughes et al. (2006) cells isolated from fragments of the livers used for transplantation
exhibited a significantly higher viability than those from livers rejected for transplantation (10 of 15
rejected livers were steatotic) [
]. Interestingly, our findings indicated that the donor livers not
suitable for transplant due to the tissue quality also had lower percentage of Alb-positive P2
cells (77.9) than it was estimated for the remaining population (85.8), p = 0.458. Thus, it turned
out that tissue quality played a major role in the successful isolation of healthy hepatocytes.
Taking into account that donors group is not representative of the patients' population (only
males) we did not include it into the further statistical analysis.
In order to find the optimal source of human hepatocytes we have focused on the method
that would enable us to estimate biosynthetic capacity of the isolated cells. We have developed
a fast and reliable technique, based on flow cytometry, to analyze production of hepatocytes'
proteins within few hours after isolation. The method to estimate quality of the hepatocytes by
the FACS analysis of the percentage of the Alb-positive cells in the P2 population is presented
here for the first time. The idea is based on the assumption that using the given antibody one
can detect target protein present in the cell in amounts above some threshold and that only
certain levels of the albumin synthesis, characteristic of active hepatocytes, allow their
detection. As it was shown for the parameters of cells isolation, patients' characteristics such as age
and chemotherapy, analyzed in the entire population individually, did not reveal statistically
significant differences in the number of active hepatocytes (percentage of the Alb- and
A1ATpossitive cells in the P2 population) in studied groups. On the other hand, the sex variable gave
us satisfying statistical significance (p = 0.038) when cells were labeled with anti-Alb antibody.
To our best knowledge, this report is the first to indicate, using FACS, that human male
hepatocytes may be more productive in terms of albumin synthesis. Generally, we have found the
anti-A1AT antibody less useful for our purposes. The reason for this could be explained by
the characteristic of the particular antibody we have used for hepatocytes' staining or by the
differential expression pattern of these two proteins. Nevertheless, albumin is the major blood
11 / 14
protein product of the liver. Difficulties in obtaining statistically significant differences
between studied groups result from the relatively low size of the groups in combination with
the broad range of the acquired data. Such a situation is typical for biological objects and more
data are required to draw far-reaching conclusions. Nevertheless, based on the Alb staining,
our results certainly indicated (p-values lower than 0.05) that: 1. samples obtained from males
gave a greater percentage of active hepatocytes than those from females and 2. specimens from
the males after chemotherapy greater than those from the treated females. In a large study on
the cytochrome P450 enzyme activity in human liver microsomes (n = 142) and its inducibility
in cultured human hepatocytes (n = 64) Parkinson et al. (2004) concluded that gender, age,
ethnicity, and liver cirrhosis of the donor should not influence the selection of human liver
samples for routine studies of drug metabolism and enzyme induction in vitro. The results
indicated that the CYP enzyme activity varied considerably from one sample to the next and
this variation was observed in human liver samples from males and females, young,
middleaged and elderly donors, and in livers from people of different ethnicity. A lack of statistical
significance was apparent between analyzed groups (with some exceptions), yet the authors
did not use combinations of these parameters for comparison [
]. In our opinion, regardless
of the differences in studied hepatocytes' activity markers, our analytical approach gives us a
deeper insight into the problem. The results obtained in our study suggest that
chemotherapeutic agents might affect albumin synthesis in male and female hepatocytes in a different
manner. There is some evidence from clinical studies that physiological variables between
human male and female hepatocytes are responsible for differential responses to drugs. In a
more recent published study male and female hepatocytes were compared in terms of their
susceptibility to drug-related hepatotoxicity and overall higher sensitivity of female primary
hepatocytes was shown [
]. There is a large body of evidence that also age of the patient
negatively affects physiological capacity of the hepatocytes. Aging impairs liver regeneration with
respect to the number of hepatocytes that are able to enter S-phase after partial hepatectomy. It
was shown that age-related hepatocyte telomere shortening might be responsible for this
decline in cell proliferation (see [
] for a review). However, in our study this variable had no
impact on the percentage of Alb-positive hepatocytes, even if we rearranged age ranges or
performed multivariate analysis. Thus, the lowered proliferative capacity of the aged hepatocytes
does not necessarily correspond to their biosynthetic potential.
In conclusion, our results show that there exists a complex dependence between the
patient's sex, chemotherapy treatment and the quality of the isolated hepatocytes in terms of
their possible usefulness in a cell-based therapy and research. Both: females and males, older
and younger people as well as patients who have undergone chemotherapy are all good sources
of viable liver cells. On the other hand, livers that are not suitable for transplant are also not
desirable in liver cells isolation. The age and the chemotherapy, when analyzed individually,
did not affect the biosynthetic capacity of the hepatocytes (Alb and A1AT production). Some
differences between groups were visible when certain combinations of these parameters were
used. The most pronounced differences (and statistically significant) were observed between:
males and females, and males and females after chemotherapy. The low number of samples is
a major limitation of our report. For this reason, we have applied very restrictive statistical
analysis, including tests for the correction for the variations of the sample size (see: Patients
and Methods section), that allowed us to draw careful conclusions. Moreover, due to the low
accessibility of such specimens for a research, many other papers report similar sizes of the
analyzed samples or sizes of the same order of magnitude [4,6,11±13].
Although more samples are needed to draw firm conclusions we ranked sources of active
hepatocytes by the median percentage of Alb-positive cells in the P2 population as follows:
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Men under 60 years old (n = 7)± 92.6 (81.2±98.7)
Men over 60 years old (n = 11)± 85.8 (59.9±97.5)
Women under 60 years old (n = 11)± 80.3 (59.9±97.5)
Women over 60 years old (n = 6)± 79.8 (56.5±95.9)
S1 Fig. Interaction plot for term "Sex/Chemotherapy".
Conceptualization: Karolina Ewa Zakrzewska, Anna Samluk, Krzysztof Dariusz Pluta.
Data curation: Karolina Ewa Zakrzewska, Anna Samluk, Agnieszka Wencel.
Formal analysis: Dorota Genowefa Pijanowska, Krzysztof Dariusz Pluta.
Funding acquisition: Dorota Genowefa Pijanowska, Krzysztof Dariusz Pluta.
Investigation: Karolina Ewa Zakrzewska, Anna Samluk, Agnieszka Wencel, Krzysztof Dudek.
Methodology: Karolina Ewa Zakrzewska, Anna Samluk, Krzysztof Dariusz Pluta.
Project administration: Krzysztof Dariusz Pluta.
Resources: Krzysztof Dudek, Dorota Genowefa Pijanowska, Krzysztof Dariusz Pluta.
Supervision: Dorota Genowefa Pijanowska, Krzysztof Dariusz Pluta.
Validation: Karolina Ewa Zakrzewska, Anna Samluk, Agnieszka Wencel, Krzysztof Dariusz
Visualization: Karolina Ewa Zakrzewska, Agnieszka Wencel.
Writing ± original draft: Krzysztof Dariusz Pluta.
Writing ± review & editing: Karolina Ewa Zakrzewska, Anna Samluk, Agnieszka Wencel,
Krzysztof Dudek, Dorota Genowefa Pijanowska, Krzysztof Dariusz Pluta.
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