Transplantation of primary human hepatocytes is a promising approach for treating certain liver diseases like metabolic disorders as well as chronic or acute liver failure. Hepatocyte transplantation is based on the application of cells in suspension. The preparation of primary human hepatocytes for cell transplantation requires either isolation from freshly resected liver tissue, thawing of cryopreserved cells, or resuspension of temporary cultivated cells. Applying the cells via the portal vein, the splenic artery, or into the splenic parenchyma can lead to reorganisation in the spleen (hepatisation) or integration into the liver parenchyma. However, a method for monitoring the processes during and following hepatocyte transplantation is still lacking. In clinical trials of hepatocyte transplantation, either biopsies were taken from the target organ or donor hepatocytes were visualised by radioisotope imaging. Both methods show limitations regarding the safety for the patient and the long-term analysis of the transplanted cells and cannot fully address the concern of distinct localisation of the cells. Recent studies have shown that magnetic resonance imaging (MRI) might be a suitable option to solve these problems. MRI enables the non-invasive assessment and visualisation of anatomy with very high spatial resolution and excellent soft tissue contrast. Compared to other non-invasive visualisation strategies such as computer tomography or scintigraphy, MRI requires no gamma- or x-ray exposition. Therefore, this method provides advanced safety to the patient, enables real-time and repetitive examinations as well as intra-operative cell tracking.
Most strategies for MRI of single cells are based on labelling with superparamagnetic iron oxide particles. These particles are commercially available in different sizes and some are already approved for clinical use. Experiments on cell labelling using nano-sized superparamagnetic iron oxide particles (SPIO) have shown their detectability after in vivo accumulation by clinical MR equipment. Recently, the first experiences with in vitro MRI of primary human hepatocytes using SPIOs have been reported. Although SPIOs are widely used for cell imaging, it has to be considered that large numbers of nano-sized particles must be incorporated into the targeted cells to enable their detection by MRI. Further limitations are the slow incorporation of unmodified SPIOs and their instability, which can cause a loss of detection and cytotoxicity. In order to achieve fast labelling, high safety of the particle load and the detectability of labelled cells on a single cell level, micron sized iron oxide particles (MPIO) were introduced to cellular imaging. Various cell types have been labelled with these particles, including macrophages and different human tumour cell lines, clearly proving the detectability of MPIO-labelled cells on a single cell level. However, the feasibility of using MPIO-labelled primary human hepatocytes for cell transplantation has not yet been evaluated.

For more detailed information please refer to the publications (J Cell Mol Med. 2008 Apr 9. [Epub ahead of print] and Int J Artif Organs. 2008 ; 31[3]:252-257).

Figure 1: Concentration and time dependency of particle uptake. (a) 18 hour incubation of primary human hepatocytes (24 hour preculture period) with increasing concentrations of MPIOs (10, 20, 30, and 40 particles/cell) resulted in an increase of particle uptake and labelling efficiency (80% ± 7.33%; 94% ± 2.79%; 96% ± 1.06%; 100%). (b) Time dependency of the particle uptake was investigated at a concentration of 30 MPIOs/cell (18 hours pre-culture period). Labelling efficiency: 83% ± 5.48%; 98% ± 1.85%; 99% ± 0.76%; 100%. Data are given as mean ± SEM.

Primary human hepatocytes - Isolation and culture conditions
Following ethical and institutional guidelines and after informed consent of tissue donors, samples were collected from a total of 13 patients undergoing partial hepatectomy (mean age of donor: 52 ± 5.8 years). Specimens (20-40g) were taken from the resected liver tissue and transferred to the laboratory under sterile conditions. Primary human hepatocytes were isolated using a modified two-step collagenase perfusion technique as already described by Katenz et al. [25]. Following the isolation procedure, cell counts and viability were determined via the Trypan blue exclusion test.
Freshly isolated hepatocytes were seeded on collagen-coated 6- and 96-well culture plates (Sarstedt, Nürnbrecht, Germany) and 8-well culture slides (BioCoat, Bedford, MA, USA) at a concentration of 1x106, 0.05x106 and 0.2x106 viable cells. Cells were cultivated in Williams´ medium E (Biochrom AG, Berlin, Germany), supplemented with 1µM insulin, 1µm dexamethason/fortecortin, 100U/ml penicillin, 100µg/ml streptomycin, 1mM sodium pyruvate, 15mM N-2-hydroxyethylpiperazine-N-´2-ethane sulfonic acid buffer (HEPES), 4mM L-glutamine and 10% fetal calf serum (FCS). After attachment phase, cells were washed with Phosphate-buffered saline (PBS; PAA, Pasching, Germany) and supplied with fresh medium. The medium was changed every 24 hours and the supernatant was collected and stored (-80°C) for further analysis.
For resuspension, primary human hepatocytes in 6-well plates were used. Cells were washed and detached from the culture plates using 0.25/0.02% Trypsin/EDTA solution (Biochrom AG). The cell suspension was collected and cell counts and viability were determined. Total recovery of resuspended cells was calculated: number of living cells after resuspension x100/number of initially seeded living cells. For reculture, again 1x106 and 0.05x106 living cells per well were seeded into 6- and 96-well plates, respectively.

Concentration and time dependency of particle uptake
The dose-determining experiments clearly revealed the dependency of the particle uptake on the concentration of MPIOs during incubation. When hepatocytes were incubated with increasing concentrations of MPIOs for 18 hours, the number of particles per cell increased from 10 to 25 (Fig. 1a). The labelling efficiency showed similar characteristics (79.50% ± 7.33% to 100%). As assessed by light microscopy, the particles were distributed throughout the entire cytoplasm of the cells and partially formed clusters of about three to five particles (Fig. 2). The incubation with MPIOs for 2 to eight 8 hours at a concentration of 30 particles/cell showed a dependency of both particle uptake and labelling efficiency on the incubation time. After 18 hours of preculture, the particle load increased from 12 to 22, while the labelling efficiency reached a plateau of approximately 100% after 4 hours of incubation (Fig. 1b). The preculture period affected the particle uptake and labelling efficiency: When hepatocytes were incubated with 30 particles/cell after being allowed to attach for 24 hours, a mean uptake of 18 particles/cell and a labelling efficiency of 96.25% ± 1.06% was achieved. The same uptake and a labelling efficiency of 98.15% ± 1.85% was reached by cells precultured for 18 hours and incubated for 4 hours with the same amount of particles.
Fluorescence microscopy revealed the intracellular localisation of the particles and showed no morphological alternations due to particle uptake (Fig. 2a-d). Electron microscopy confirmed the incorporation of the particles both as single particles and as clusters. The encasement of the particles within cytoplasmic vesicles as well as their iron core was clearly detectable (Fig. 2e).
In order to investigate the number of particles per cell required to enable detection by magnetic resonance instrumentation, samples of hepatocytes with increasing particle content embedded in agarose were analysed. Signal extinctions of labelled cells correlated to the number of incorporated particles and increased with the particle load. Images of cells containing 18 ± 1 or 25 ± 2 particles displayed distinct signal extinctions at low SNR (28.82 ± 5.27 and 30.66 ± 5.37) that clearly contrast against the high image background caused by the aqueous agarose medium. On the basis of previously published MR images of MPIO-labelled cells in agarose suspension, an uptake of about 17 – 20 particles per cell (Fig. 3a, b) was assumed to be sufficient for cell detection with MR equipment at 3.0 Tesla. This particle load was achieved by incubating the hepatocytes with 30 particles/cell. Agarose samples containing cells with 10 ± 2 or 16 ± 1 particles per cell showed lower signal extinctions at higher SNR (45.09 ± 8.41 and 38.38 ± 7.12) and were therefore estimated as not suitable for cell detection in agarose samples. MR images of agarose samples containing an identical number of unlabelled cells (Fig. 3c, d) or the equivalent amount of MPIOs without cells (Fig. 3e, f) had a uniform appearance and revealed no distinct signal extinctions. According to these data the concentration of 30 particles/cell and an incubation time of four hours resulting in a mean particle load of 18 were defined as the standard conditions for further cell labelling experiments.

Figure 3: Cells labelled with 18 ± 1 MPIOs/cell were clearly detectable both in sagittal and axial slices (a, b) at a concentration of 1000 cells/250µl. The same number of non-labelled cells (c, d) or a correlating number of MPIOs suspended in agarose (e, f) showed no detectable distinct signal changes.

Particle uptake and retention
After incubation of cells with MPIOs for four hours, 97.34 ± 0.7% of the cells were labelled with an average of 18 ± 1 particles per cell (Fig. 6d). 24 hours after labelling, the mean particle load of adherent (group B) and resuspended hepatocytes (group D) was 19 ± 1 and 18 ± 1, respectively. At the end of the culture period, the cells of group B contained an average of 19 ± 1 and group D 18 ± 1 particles. There were no statistically significant differences in the particle load between the adherent group and the resuspension group during the entire culture period. Cell viability of freshly isolated primary human hepatocytes was 75.22% ± 2.24%. By trypsin treatment, 52.17% ± 3.79% of unlabelled (group C) and 55.11% ± 6.75% of labelled (group D) initially seeded viable hepatocytes could be resuspended, achieving a viability of 68.93% ± 3.24% and 72.53% ± 3.54%, respectively. There was no evidence that the particles had a negative effect on the success of the trypsin treatment. Freshly isolated cells showed the typical morphological appearance of primary human hepatocytes (Fig. 2a, b). They presented a polygonal shape, granular cytoplasm with vesicular inclusions and one or more nuclei. The labelling procedure caused no alternations of the morphology of hepatocytes (Fig. 2c, d). Shortly after incubation, the particles were situated on the cell membrane, at later time points they were detected mostly in the peri-nuclear cytoplasm, both as single particles as well as clusters. Over the entire culture period, no morphological differences were seen between the four groups.

Cell integrity and metabolism
The mitochondrial activity of resuspended cells was not affected by the particle incorporation. Statistical significant differences were not detected in the resuspension groups between unlabelled and labelled cells at culture day 2 (group C: 2.51 ± 0.10, group D: 2.32 ± 0.33) and culture day 6 (group C: 2.28 ± 0.27, group D: 2.60 ± 0.30). No statistical differences were detected among the groups concerning the total protein at the end of the culture period verifying the same amount of cells in the different groups (data not shown). In both resuspension groups, a peak of AST release was observed 24 hours after trypsin treatment, followed by a rapid decrease from 106.8U/L (group C) and 113.4U/L (group D) to 36U/L and 38.4U/L on the next day, respectively (Fig. 4a). In the meantime, AST levels of the adherent groups decreased from 49.5U/L (group A) and 48.2U/L (group B) to 27.7U/L and 28.7U/L, respectively. Throughout the rest of the culture period, the AST release of all groups was constant. LDH concentrations of both resuspension groups increased between day 2 and day 3 to the levels of the control groups (C: 11.2U/L to 14.6U/L; D: 9.7U/L to 14.8U/L), while the other groups showed no alternations. From day 3 to the end of the culture period there were no significant differences between the groups concerning the LDH release. A significant increase of the LDH release could only be observed in the resuspension groups from day 5 to day 6. Concerning the particle load, no differences in the cell damage parameters were detected between labelled and unlabelled cells from day 2 to day 6. Urea synthesis of resuspended hepatocytes was significantly lower 24 hours after resuspension, but showed a rapid increase thereafter (Fig. 4b). From day 3 until day 6, the mean urea synthesis of the groups varied between 1.4mmol/l and 1.8mmol/l per day and showed no significant differences among the groups. Albumin secretion increased during the culture period in all groups and did not show significant differences within the four groups on any time point of measurement (Fig. 4c), but the albumin production of the group C tended to be higher than group D at each sample point. Differences between the labelled and unlabelled cells concerning their metabolic activity were not detectable between day 2 and day 6.

This is the first study on preparation of MPIO-labelled primary human hepatocytes. The feasibility of the intracellular incorporation of the particles as well as the detection of labelled primary human hepatocytes by clinical MR equipment could be shown in vitro. MPIO-labelled cells could serve as a valuable tool for both basic research in cell based regenerative therapies and for quality control in the clinical setting of human hepatocyte transplantation.