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. 2010 Nov 5;1(2):81–92. doi: 10.3727/215517910X536627

Transplant of Primary Human Hepatocytes Cocultured With Bone Marrow Stromal Cells to SCID Alb-uPA Mice

S A Mohajerani *, M Nourbakhsh *, A Cadili *, J R Lakey †, N M Kneteman *
PMCID: PMC4776167  PMID: 26966632

Abstract

Hepatocytes are vulnerable to loss of function and viability in culture. Modified culture methods have been applied to maintain their functional status. Heterotypic interactions between hepatocytes and nonparenchymal neighbors in liver milieu are thought to modulate cell differentiation. Cocultivation of hepatocyte with various cell types has been applied to mimic the hepatic environment. Bone marrow stromal cells (BMSC) are plastic cell lines capable of transforming to other cell types. In this study hepatocyte coculture with BMSCs achieved long-term function of human hepatocytes in culture for 4 weeks. In vitro functional status of human hepatocytes in BMSC coculture was compared with fibroblast coculture and collagen culture by measuring albumin, human-α-1-antitrypsin (hAAT), urea secretion, CYP450 activity, and staining for intracellular albumin and glycogen. After 2 weeks in culture hepatocytes were retrieved and transplanted to severe combined immunodeficiency/albumin linked-urokinase type plasminogen activator (SCID Alb-uPA) mice and engraft-ment capacity was analyzed by human hepatic-specific function measured by hAAT levels in mouse serum, and Alu staining of mouse liver for human hepatocytes. Hepatocytes from BMSC coculture had significantly higher albumin, hAAT secretion, urea production, and cytochrome P450 (CYP450) activity than other culture groups. Staining confirmed the higher functional status in BMSC coculture. Transplantation of hepatocytes detached from BMSC cocultures showed significantly higher engraftment function than hepatocytes from other culture groups measured by hAAT levels in mouse serum. In conclusion, BMSC coculture has excellent potential for hepatocyte function preservation in vitro and in vivo after transplant. It is possible to use BMSC hepatocyte coculture as a supply of cell therapy in liver disease.

Key words: Human hepatocytes, Bone marrow stromal cells, In vitro function, SCID-uPA mouse, In vivo function

INTRODUCTION

Developing an effective system for the long-term culture of primary human hepatocytes would be of tremendous benefit in clinical, pharmaceutical, and tissue engineering studies and applications. The availability of a practical system for long-term functional human hepatocyte culture would greatly enhance research in the areas of viral hepatitis and antiviral drug development, drug hepatotoxicity, as well as development of effective bio-artificial liver (BAL) systems (37). Other sources of human hepatocytes such as immortalized human hepatocytes (42), stem cell-derived hepatocytes, and fetal human hepatoblasts have not shown great promise so far as their functional status is much lower than primary hepatocytes.

In line with such goals, researchers have tried different culturing methods to enhance the ability of human hepatocytes to maintain their viability and function in vitro. Based on the assumption that the extracellular matrix is an important modulator of cell polarity and function of human hepatocytes (10), attempts have been made to culture human hepatocytes on 3D culture mediums such as collagen sandwich or algenate encapsulation (13,22,24) or 2D cultures such as collagen or Matrigel (26,27) without adding much complexity to the culture system (31).

Heterotypic interactions between human hepatocytes and nonparenchymal neighbors in liver milieu have been reported to modulate cell growth and differentiation (2). According to that theory, cocultivation of hepatocytes and nonparenchymal cells like fibroblasts, liver epithelial cells (3), and endothelial cells (38) has been used to stabilize hepatocyte phenotype. None of these culture systems, however, has been demonstrated to support hepatocytes’ function to the extent that they can still retain their function for transplantation (35). In this study, we report on the results of extending a novel culturing system for human hepatocytes that relies on coculturing the hepatocytes with bone marrow stromal cells (BMSCs) with application for transplant of human hepatocytes. The rationale behind choosing BMSCs was that they have the ability to differentiate into various kinds of cells (41,43), including the mesenchymal (29) and epithelial cells (30), whick could support hepatocyte survival and function. BMSCs also have the capacity to differentiate into hepatocytes (17,40,44). Also, it has been shown that BMSCs could enhance proliferation and support differentiation of rat hepatocyte in culture (25).

In this study, we compared the BMSC hepatocyte coculture with two other conventional methods of culturing hepatocytes (collagen culture and fibroblast coculture). We compared the culture systems with regard to the long-term viability and function of the cultured human hepatocytes. In addition, we retrieved the human hepatocytes from the culture dishes after 14 days in culture and transplanted them into severe combined immunodeficiency/albumin linked-urokinase type plasminogen activator (SCID Alb-uPA) mice to assess the function of the cells in vivo. The human hepatocytes cultured with BMSCs showed higher levels of function in vitro compared with the other groups; also, after transplantation, the BMSC cocultured group displayed significantly greater retention of their functional capacity in vivo.

MATERIALS AND METHODS

Cell Isolation and Culture Conditions

For human subjects used in this study ethical approval for study protocol was obtained from the University of Alberta Health Research Ethics Board and proper informed consent in writing was obtained from all patient donors. Human hepatocyte were isolated from segments of human liver tissue obtained from resection specimens flushed with Liberase HI (Roche, IN) and digested according to modified method by Seglen (33). The cell pellet was resuspended in DMEM (Invitrogen, Ont.) supplemented with 1 nM insulin and 1 nM dexa-methasone (Sigma, Oakland, Ont.). Viability of freshly isolated human hepatocytes was determined by trypan blue.

BMSCs were isolated according to the protocol previously described (45). Bone marrow was collected from hip bone marrow obtained from patients that had undergone total hip replacement. After filtration and density gradient centrifugation the mononuclear cell phase was collected. Cells were seeded in culture flasks and nonad-herent cells were removed after 48 h. The adherent cells were expanded, passaged, and cultured. Phenotypic characterization confirmed that the cells are BMSCs. Human BMSCs were expanded by culturing in growth medium [DMEM, 1% insulin-transferrin-selenium (GIBCO), 5 μg/ml linoleic acid, 100 μM ascorbic acid 2-phosphate, 1 nM dexamethasome, 10 ng/ml EGF (all Sigma), 100 U/ml penicillin/streptomycin, and 2% fetal calf serum (all Invitrogen)] and stored by cryofreezing.

The coculture systems were set up the day prior to culturing the human hepatocytes by seeding 2 × 105/100-mm plates of nontreated human BMSCs for BMSC coculture group and commercially available mitomycin C-treated primary mouse embryo fibroblasts (Millipore, Etobicoke, ON) for fibroblast coculture group. We prepared collagen culture dishes by coating with 25 μg/cm2 type I collagen (0.1 mg/ml) according to the previously described method (6). Freshly isolated human hepatocytes were seeded at a density of 1 million viable cells/100-mm dishes in three different groups: collagen-coated culture (CC), fibroblast coculture (FC), and BMSC coculture. The medium consisted of low-glucose DMEM with 10% fetal calf serum, penicillin, and streptomycin (100 IU/ml) (Invitrogen, Ont.). After 4 h, the medium was changed to remove the unattached cells from the cell culture. Cultures were in 37°C incubators in 95% air/5% CO2 for 28 days. Thereafter, the medium was changed every 48 h.

Hepatocyte Function Characterization

We prepared culture slides on day 28 by using chamber polystyrene vessel tissue culture-treated glass slides (Becton Dickinson, Ont.). We used immunohistochemistry staining for intracellular albumin. After quenching and blocking, slides were incubated with rabbit anti-human serum albumin (Abcam, Cambridge, MA, USA) followed by biotinylated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, PA, USA). After incubation with the avidin-biotin complex (ABC)-enzyme complex, they were developed using diaminobenzidine as chromagen (Vector Laboratories, CA, USA) and then counterstained with Harris hematoxylin and alcoholic eosin Y. We also evaluated the hepatocyte ability to synthesize cytosolic glycogen by periodic acid-schiff (PAS) staining. Culture dishes containing the hepatocytes were fixed in 95% alcohols for 10 min. Samples were then oxidized in 1% periodic acid, and then rinsed and treated with Schiff’s reagent. Alu staining was performed to detect engrafted human hepatocytes in mouse liver after transplant. In brief, sections were covered with human Alu-DNA-Probe (PR-1001-01, Innogenex, San Ramon, CA) for overnight at 37°C. Then, posthybridization washes and detection were performed according to the protocol provided with the Super Sensitive™ Polymer-HRP ISH detection kit (DF300-YCX, BioGenex Inc., San Ramon, CA, USA). Finally, probes were developed by DAB chromagen as dark brown compared to background blue of mouse cells nucleus. All photographs are taken with the same magnification.

Hepatocytes Function

Cell viability in culture was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) viability assay kit (Biotium, CA, USA) according to the manufacturer’s protocol. Viability was measured as percentages of sample to control (hepatoma cell line). Human hepatocyte culture in three different culture systems (BMSC and FC coculture, and CC) was assigned to 100% control group (hepatoma cell line). For coculture groups (BMSC and FC) we used BMSCs and fibroblast alone (without hepatocytes) as background. In order to normalize in vitro data based on viability, we used the same number of viable HH for each culture experiments (per 106 viable cells).

Albumin secretion in media was measured by sandwich enzyme-linked immunosorbent assay (ELISA) by using goat anti-human albumin antibody affinity purified as primary antibody and goat anti-human albumin cross adsorbed polyclonal antibody, HRP conjugated as secondary antibody (Bethyl Laboratories, Montgomery, TX, USA) according to the manufacturer’s protocol. At each time point the cultured cells were washed twice with serum-free medium and then incubated in serum-free medium for 24 h after which the tissue culture supernatant was gathered from culture plates and stored at −80°C until analysis. We used a BMSC culture alone without human hepatocytes as negative control. The data for all in vitro function tests were normalized for 106 cells per culture dishes in different study groups.

Human α-1-antitrypsin (hAAT) secretion (ng/ml/day/106) in culture was analyzed by a method previously described (14). Samples of primary human hepatocyte culture supernatant analyzed by sandwich ELISA using a polyclonal goat anti-hAAT antibody (#81902, Diasorin, Stillwater, MN, USA) as the capturing antibody. A portion of the same antibody was cross-linked to horseradish peroxidase (#31489, Pierce, Rockford, IL, USA) and used as the secondary antibody, with signal detection by 3,3,5,5-tetramethylbenzidine (Sigma, St. Louis, MO, USA).

To evaluate hepatocyte-mediated biotransformation of ammonia to urea, seeded hepatocytes were exposed to 10 mM/L NH4Cl in culture medium. Samples of media were collected at the beginning and after 24 h of exposure to ammonia. Urea concentration was measured colorimetrically using the urea nitrogen reagent set (Bio-Tron Diagnostics, Hemet, CA, USA) and an ELISA reader at 540 nm. Concentrations of urea were determined using a standard curve of urea (0–45 μmol/ml) according to the manufacturer.

Cytochrome P450 (CYP1A1) enzymatic activity was measured by quantifying the amount of resorufin produced from the CYP-mediated cleavage of ethoxyresorufin O-deethylase (EROD) as described previously (4). EROD (8 μM) was incubated with cell cultures for 30 min, medium was collected, and resorufin fluorescence was quantified at 530/590 nm (excitation/emission wavelengths).

In Vivo Transplantation and Study

Cultured human hepatocytes in the three culture groups were recovered after 14 days in culture and transplanted into SCID Alb-uPA mice. This was performed in order to evaluate their capacity for in vivo function after retrieval from in vitro culturing. We also transplanted freshly isolated human hepatocytes as a control for each experiment. SCID Alb-uPA mice are homozygous for the severe combined immune deficiency (SCID) trait. This renders them virtually deficient in T-cell and B-cell function, thus making them good recipients of xenografts. They also express a urokinase-type plasminogen activator (uPA) gene under the control of an albumin promoter. This leads to extensive liver toxicity and creates an environment that is ideal for the xenotransplantation and subsequent expansion of human hepatocytes (23).

After 14 days in culture, cells from each of the three different culturing groups were trypsinized, and after determining the viability by trypan blue, were washed and resuspended in serum-free media. The recovered cells from culture dishes included human hepatocytes and their feeder layers, BMSCs or mitomycinC-treated fibroblasts as we did not separate them. The recovered cells were subjected to flow cytometric analysis using FACS Calibur (Becton Dickinson Co., Mountain View, CA, USA) to assess the albumin-positive cell population. This was performed to normalize the number of transplanted cells from each group to albumin-positive cell population recovering from culture dishes. Cells were labeled with goat anti-human albumin Ab-FITC conjugated (Bethyl Laboratories, Montgomery, TX, USA) and analyzed by flow cytometry. We used saponin to enable membrane penetration. The percentage of albumin-positive cells and the mean fluorescent intensity of the positive cells were determined after compensating for auto-fluorescence of hepatocytes (100–101). This was done using BMSC cell lines as negative controls and freshly isolated hepatocytes as positive controls. Data were analyzed using Cell Quest software (Becton Dickinson). After this, all the recovered cells from the dishes that included hepatocytes plus their feeder layers, BMSC or mitomycin C-treated fibroblasts, were transplanted into 10–14-day-old SCID Alb-uPA mice by injection into the inferior pole of the spleen. Twenty mice were transplanted in each of the groups. After transplant, two blood samples, initially at 6 weeks and then 8 weeks posttransplant, were obtained from each mouse to measure the hAAT level by ELISA. Serum hAAT level determination was used as a marker for graft survival and function of hepatocytes in the transplanted mice by methods described previously (14). The lower limit of the linear part of the hAAT ELISA assay is 3.125 μg/ml.

Statistics and Data Analysis

Quantitative in vitro results were obtained from five experimental repeats using hepatocytes from five different isolations. For each of these five experimental repeats, we used three culture dishes at each time point for each group. Each value represented the mean + SD. One-way repeated measures analysis of variance (ANOVA) followed by the Tukey post hoc test multiple group comparison was used to analyze group differences of the resultant data. The threshold for statistical significance was considered p < 0.05.

RESULTS

Staining for Intracellular Albumin and Glycogen

Intracellular human albumin staining was strongly positive in BMSC coculture cells at day 28 (Fig. 1). Although we did not count the number of positive cells, hepatocytes cocultured in FC or cultured on CC revealed only sparse, weak, and scattered staining for human albumin at day 28. The negative control of BMSC culture alone (without hepatocytes) staining was negative. The positive control for this experiment was section of fresh human liver tissue.

Figure 1.

Figure 1

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Immunohistochemical staining of intracellular albumin in cultured human hepatoctes on day 28. Positive staining of albumin as dark brown staining (arrow) was evidenced in hepatocyte co-cultured with BMSCs (A). However, the fibroblast coculture (B) and collagen culture (C) showed weak staining for intracellular albumin. The control negative (BMSCs without hepatocytes) albumin staining caused no brown coloration (D). Human liver section used as positive control (E). Intense positive staining of albumin was observed in cell pellets of human hepatocytes detached from BMSCs on day 14 before transplant (F). Original magnification: 200×. Scale bars: 200 μm.

Positively stained glycogen granules, which exhibited a dark purple-magenta color, were detected in the cytoplasm of human hepatocytes cocultured with BMSCs at day 28 of culture, whereas BMSCs alone (no hepatocytes) as a negative control showed no pink coloration (Fig. 2). The CC and FC hepatocytes showed only weakly positive staining at day 28. The positive control used in this case was sections of fresh human liver tissue section.

Figure 2.

Figure 2

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Periodic acid-Schiff (PAS) staining of intracellular glycogen storage in cultured human hepatocytes on day 28. Dark purple-magenta color (arrow) of glycogen was positive in BMSC coculture (A) but weak in fibroblast cocultured (B) and collagen culture (C). The human liver section was used as control positive with strong color (D). Original magnification: 200×. Scale bars: 200 μm.

Hepatocytes Functions In Vitro

By using MTT viability test we showed that human hepatocytes lose their viability gradually and do not grow in culture groups. The gradual decrease in viability was observed in human hepatocytes cultured in all three different groups. In BMSC and FC the viability was not significantly different in culture time points, but CC was significantly lower after day 21 (p < 0.05) (Fig. 3).

Figure 3.

Figure 3

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The cell viability during time line in culture measured by the MTT assay is shown as percentages of sample to control (hepatoma cell line) (A). Albumin secretion in media by human hepatocytes cultured in different groups during 28 days in culture (B). The hepatocyte/BMSC coculture had significantly higher albumin secretion than the hepatocyte/fibroblast coculture (FC) and collagen culture (CC) on day 21 and 28 (*p < 0.05). The albumin secretion in BMSCs alone as control negative was zero. The human α-1-antitrypsin (hAAT) secretion in culture media by human hepatocytes in different culture groups (C). The hepatocytes/ BMSC group showed significantly higher secretion of hAAT in culture in compare to other groups on day 21 and 28 (*p < 0.05).

The albumin secretion into the media was not significantly different at day 1 of culturing among the groups (Fig. 3). Hepatocytes in the BMSC coculture group maintained albumin secretion at a plateau level from day 7 (40.5 ± 3.8 ng/ml/day/106 cells) until day 28 (34.3 ± 2.4 ng/ml/day/106 cells). Albumin secretion from BMSC cocultured human hepatocytes was significantly higher than other groups (p < 0.05, n = 5) on days 21 and 28. The negative control of BMSC alone resulted in zero albumin secretion at all time points.

Although hAAT secretion level in BMSC dropped from 2230 ± 120 ng/ml/day/106 cells on day 1 to 1200 ± 110 ng/ml/day/106 cells on day 28, the secretion of hAAT in BMSC cocultures was significantly higher than the other culture groups on day 21 and 28 (p < 0.05, n = 5) (Fig. 3). The negative control of BMSCs alone (without hepatocytes) was negative all the time points.

There was a significant difference in urea concentration between BMSC (47 ± 5.3 μmol/ml/day/106 cells) and FC group (25 ± 3.5 μmol/ml/day/106 cells) on day 14 (p < 0.05) and thereafter. The urea concentration in collagen culture was significantly lower than in the BMSC group on day 14 and after (p < 0.05). To eliminate the effect of cocultured cells on the values in BMSC and FC group, we measured the urea production by BMSCs and fibroblasts alone (without hepatocytes) and used as background (Fig. 4).

Figure 4.

Figure 4

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Urea synthesis as a marker of human hepatocyte function in different culture groups during 28 days in culture (A). The hepatocytes cultured on BMSC had a significantly higher level of urea synthesis than fibroblast coculture (FC) and collagen culture (CC) on day 14 and after. Time course of ethoxyresorufin O-deethylase (EROD) activity in different groups of culture (B). Cultures were incubated for 30 min with 7-ethoxyresorufin, and the resorufin formed was evaluated by fluorescence described in Materials and Methods. The resorufin formed was significantly higher in BMSC culture group on day 28 in culture than fibroblast coculture and collagen culture (*p < 0.05).

Hepatocytes in the BMSC coculture group exhibited a statistically significant higher level of EROD activity (14.9 ± 3.3 pmol/min/106 cells) compare to other culture groups on day 28 (p < 0.05). EROD enzymatic activity of the freshly isolated hepatocyte homogenate (10.6–25.3 pmol resorufin/min/106 cells) was used as the standard for comparison. EROD activity remained relatively constant in BMSC but dropped off in others from day 14 up to day 28. EROD activity in BMSC group was approximately five times higher than the activity in CC and three times than FC on day 28 (Fig. 4).

Transplantation of Cultured Human Hepatocyte

The percentage of albumin FITC-positive cells in each group after recovery from the culture dishes (on day 14) was estimated by flow cytometry. The results of the FACS analysis for each group showed that 85.9% of the freshly isolated hepatocytes, 78.5% of the cells recovered from BMSC coculture, 76.2% from fibroblast coculture, and 82.3% from collagen culture were positive for albumin staining (Fig. 5). The freshly isolated human hepatocytes were used as positive control and BMSC cells alone (without hepatocytes) were used as negative control. Final preparation of fresh hepatocyte isolation contains cell debris and other cell types reside normally in liver tissue. Our previous studies have demonstrated that these non-hepatocyte cells do not survive and expand after transplant. Post-sort analysis of FACS-purified subpopulations revealed a hepatocyte purity of 72–82% of each subpopulation of recovered cells from the cocultures in different experiments. By using FACS numbers we transplanted 1 million albumin-positive cells to each SCID-uPA mouse in each culture group. We transplanted total of 20 mice in each group chosen from five different donors (4 mice each). Donor characteristics are listed in Table 1.

Figure 5.

Figure 5

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Fluorescein-activated cell sorting (FACS) analysis of recovered cell population from culture dishes on the basis of albumin expression. Flow cytometry analysis for the assessment of number of albumin-positive cells after detachment from culture dishes on day 14 before transplanting to the mice in BMSC coculture (B), fibroblast coculture (C), collagen culture (D) were used to determine the albumin-positive cells in the recovered suspension before transplantation into mice. Freshly isolated human hepatocyte used as a positive control (A) and human bone marrow stem cells alone (BMSC without hepatocytes) used as control negative (E).

Table 1.

Donor Characteristics of Liver Tissue Donors That Were Used to Isolate the Human Hepatocytes for This Study

Type of Tissue Age (Years) Sex Cold Ischemia Time (min) Fresh Viability (%) Patient’s Disease
Normal 31 F 60 81 Recurrent biliary stone
Normal 25 F 45 68 Focal nodular hyperplasia
Normal 33 F 60 81 Hemangioma
Normal 76 M 50 86 Colon cancer
Normal 45 F 60 94 Cholangiocarcinoma
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Day 14 was selected for transplantation as the in vitro data showed acceptable function of the cultured hepatocytes particularly in hAAT secretion. Transplant of freshly isolated human hepatocytes was used as control from the same donors. The function of the transplanted hepatocytes was evaluated through measurement of the hAAT level from each mouse at 6 and 8 weeks after transplantation. The results of hAAT levels in weaning mice at 6 weeks posttransplant according to experimental group are summarized in Table 2. Also, at 8 weeks posttransplant hAAT level in mice transplanted with hepatocytes from BMSC group (mean ± SD: 13.8 ± 11.7; median: 13.3; min: 5; max: 61) was significantly higher than FC (mean ± SD: 0.5 ± 0.98; median: 0; min: 0; max: 3), and CC group (mean ± SD: 0.3 ± 0.87; median: 0; min: 0; max: 3), and lower than fresh group (mean ± SD: 94 ± 62.19; median: 101; min: 27; max: 240) (p < 0.05). The Box-Whisker diagram in Figure 6A also shows that the median and the quartiles hAAT values in BMSC group are higher than FC and CC and lower than fresh group. The presence or absence of engraftment and repopulation of human hepatocyte in mouse liver was confirmed by Alu staining, which stains only for human nucleus as dark brown (Fig. 6B). Transplanted human hepatocytes were detected in the BMSC coculture group but not in FC and CC groups 12 weeks posttransplant in the background of the mouse liver (lacks dark brown nuclear staining) by Alu staining.

Table 2.

Serum Human α-1-Antitrypsin (hAAT) Level (μg/ml) in Mice Transplanted With Human Hepatocytes From Different Culture Groups Compare to Freshly Isolated Human Hepatocytes Transplanted From the Same Donors at 6 Weeks Posttransplant

Group No. of Mice hAAT = 0 (μg/ml) hAAT = 0–15 (μg/ml) hAAT > 15 (μg/ml)
Fresh 17 0 (0%) 7 (41%) 10 (59%)
Bone marrow coculture 17 0 (0%) 13 (76%) 4 (24%)
Fibroblast coculture 16 12 (75%) 4 (25%) 0 (0%)
Collagen 16 14 (87.5%) 2 (12.5%) 0 (0%)
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Figure 6.

Figure 6

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(A) Functional assessment of engrafted human hepatocytes measured by human α-1-antitrypsin (hAAT) serum values of SCID Alb-uPA mice. Box-Whisker diagram shows that median and quartiles hAAT values of mice transplanted with cultured human hepatocytes from BMSC coculture are higher than fibroblast coculture (FC) and collagen culture (CC) group, and lower than fresh at 8 weeks posttransplant (*outlier hAAT values in the graph). (B) Demonstration of mouse liver chimerism. Alu staining of chimeric mouse liver cross section after 12 weeks posttransplantation of human hepatocytes from BMSC coculture (a) shows nodules of proliferating human tissue with dark brown nuclei (arrow), surrounding by mouse tissue that lacks nuclear staining. In fibroblast coculture transplanted liver (b) no human tissue were detected. Mouse liver transplanted with fresh human hepatocytes (c) and mouse without transplant (d) as controls are shown. Regions that contain hepatocytes with positive and negative nuclei are defined as human (H) and mouse (M) areas, respectively, the boundary being indicated by a dashed line. The Alu staining was done on mouse with hAAT value of 61 in the BMSC group, 94 in the fresh group, and 0 in the FC group. Original magnification: 200×. Scale bars: 200 μm.

DISCUSSION

Primary human hepatocytes lose their normal function and viability when cultured in vitro (18), suggesting that the in vivo microenvironment (11) plays a major role in maintenance of phenotype. Extensive studies for optimization of culture conditions for human hepatocytes (16,32) indicate that heterotypic cell interaction is an essential component for maintaining long-term survival and function. Often, hepatocytes are cultured in a 2D or 3D environment that provides space support for hepatocytes (28,34), cellular aggregation (21), or cell– matrix interaction (1). Some studies have described the maintenance of hepatocyte function in vitro for more than 30 days in culture, but maintenance of hepatocyte-specific functions along with capacity to restore their proliferative and functional activity after retrieving from cultures and transplantation have not been reported in long-term cultured hepatocytes. To test functional activity of hepatocytes in vitro, both synthetic and metabolic, we used synthetic tests for albumin and hAAT and metabolic testing with urea production and CYP450 activity. The results of our studies indicated the superiority of cocultures to matrix-only cultures (like collagen) in maintaining both synthetic and metabolic activities of hepatocytes for longer term at higher level. However, using techniques to form spheroids in biomatrix cultures (like synthetic polymers) undoubtedly could enhance hepatocyte function in culture (15). Technical difficulties are a major big challenge for routine use of these systems.

The goal of any coculture system is to simulate the in vivo environment for hepatocytes. Heterotypic cell–cell interaction, in addition to the homotypic interaction, is an important modulator for hepatocyte functions (11). Normally, hepatocytes are in direct or indirect cell-to-cell contact with various cells types in liver (7), but it is not clear which cell is the mainstay of this microenvironment interaction. For our coculture system, we speculate that undifferentiated plastic cells like BMSC may provide more robust substitute for the in vivo microenvironment than differentiated cells like fibroblast or biliary epithelial cells. This could be due to adaptability of plastic cells and also secretion of different cytokines and growth factors such as interleukin-6 by BMSCs (12). BMSCs also have the advantage of providing extracellular matrix (ECM) for the hepatocytes by production of collagen, laminin, and fibronectin (8). In the current study, we describe coculture of human hepatocytes with BMSCs in order to maintain in vitro survival and function of hepatocytes. Our results revealed that the BMSC coculture system maintained hepatocyte-specific function for 4 weeks at levels that were comparable to those of freshly isolated human hepatocytes. This study confirms the efficacy of the hepatocyte–BMSC coculturing system in maintaining prolonged hepatocyte differentiation and function in vitro in comparison to fibroblast coculture. This was not different from other studies that have shown partial dedifferentiation of hepatocytes in coculture with differentiated cell lines such as fibroblasts, epithelial, or endothelial cells after 1 month in culture (19,36).

The most dramatic finding of this study was that hepatocytes retrieved from the BMSC coculture system can be more successfully transplanted into SCID Alb-uPA mice. A significant number of mice transplanted with human hepatocytes cocultured with BMSCs showed measurable hAAT levels in serum, which indicates engraftment, expansion, and function of the transplanted cells. The proportional number of successful hepatocyte transplants after 14 days in BMSC coculture was higher than two other culture groups considering the hAAT = 15 cut-off point; however, none of our BMSC coculture transplanted mice showed hAAT values over 100 μg/ml like fresh group. Long-term cultures of both human hepatocytes in collagen and cocultures with fibroblasts did not provide human hepatocytes that were capable of successful transplantation. This result indicates that the BMSC coculture can preserve the differentiation of hepatocytes over a prolonged period of time, which indicates that their capacity for subsequent in vivo function and engraftment is better than two other culture groups but lower than fresh hepatocyte transplant (5). This is probably due to the reality that plastic BMSCs could better preserve the functional status of hepatocytes in culture as depicted by our in vitro data. Moreover, BMSCs appear able to interact with their neighbor hepatocytes particularly in a damaged liver milieu like the SCID Alb-uPA mouse after cotransplantation along with hepatocytes (20). Also, cotransplantation of BMSCs and hepatocytes from a coculture appears to have a systemic and local effect on hepatocytes nested in liver by increasing serum and local cytokine levels (39), which could explain the higher graft function in the BMSC group compare to the fibroblast group. The provision of cells for transplantation would, in many ways, present the ultimate test for the success of any in vitro culturing system of human hepatocytes. A successful system could have profound and revolutionary clinical implications, as such a method would potentially allow for liver reconstitution or provide an external bioartificial liver device (BALD) in patients with liver failure. In particular, BMSCs could be obtained in advance from transplant recipients and the BMSC–hepatocyte co–cultures might be implanted back into the patient. This holds significant promise for research and clinical application involving many disease states (9). Besides, it is important to note that using any immortalized cell line like fibroblasts in a coculture might face the potential risk associated with the transmission of immortalized cells or tumorigenic products into the patient’s circulation. Therefore, primary cells like BMSCs are better candidates for the hepatocyte coculture if any clinical application is considered.

While these are very encouraging results, challenges to developing the ideal conditions for hepatocyte culture still persist. Only 24% of the mice transplanted with hepatocytes from the BMSC coculture group showed hAAT levels greater than 15 μg/ml compared to 59% of mice transplanted with fresh human hepatocytes. Although transplant success depends on many other factors, such as cell viability and donor characteristic, the lower hAAT levels in coculture-derived cells could be due to partial loss of function in culture after 14 days. Nevertheless, we believe that the application of this coculturing system represents a significant qualitative leap in the endeavor to master the isolation, preservation, and later reintegration of functional human hepatocytes into damaged liver. Future research should be directed on finding the molecular signals that mediate interactions of BMSCs and hepatocytes, which will lead us to manipulate this culture system. The mechanisms underlying enhanced hepatic function in BMSC cocultures should provide fruitful study in the future.

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