Problems with the limited availability of human hepatocytes for cell transplantation may be overcome by efficient cryopreservation techniques and formation of appropriate cell banking. In this study we investigated the effect of the disaccharide trehalose on the cryopreservation of human hepatocytes. For analysis, liver cells were frozen in culture medium containing 10% dimethyl sulfoxide (DMSO) that was supplemented with varying concentrations of trehalose. During the postthawing culture period, viability, plating efficiency, total protein, cell proliferation, enzyme leakage, albumin and urea formation, as well as phase I and II metabolism were analyzed. In the pilot study, among the concentrations investigated, 0.2 M trehalose showed the best overall outcome. Compared to the use of DMSO alone, we found significant improvement in postthaw cell viability (62.9 +/- 13 vs. 46.9 +/- 11%, P 0.01) and plating efficiency (41.5 +/- 18 vs. 17.6 +/- 13%, P 0.01) in the trehalose group. The use of trehalose as an additive for cryopreserving human hepatocytes resulted in a significantly increased total protein level in the attached cells, higher secretion of albumin and a lower aspartate aminotransferase (AST) level after thawing. In conclusion, the use of trehalose as cryoprotective agent significantly improves the outcome of human hepatocyte cryopreservation.

For more detailed information please refer to the publication (Liver Transplantation 2007, 13: 38-45).

Primary human hepatocytes constitute a suitable model for the utilization in pharmacological and toxicological studies, cell transplantation as well as in artificial extracorporeal liver support systems. Cells obtained from livers unsuitable for conventional whole organ transplantation can be used for cell transplantation; they have the advantage of being less invasive and less expensive. However, the availability of human hepatocytes is limited, and the maintenance of primary cultures is possible for only a few weeks. Successful long-term preservation techniques are therefore needed to provide a sufficient amount of human liver cells for in vitro experiments and, eventually, for the treatment of patients. Cryopreservation represents one of the useful options for these purposes. In the past few years, several groups have developed and optimized the cryopreservation of freshly isolated hepatocytes from different species. Although progress has been reported, problems such as a significant decline in the post-thaw cell viability, poor attachment and limited function of the thawed cells still persist. Most protocols for the cryopreservation of liver cells are based on the combined use of dimethyl sulfoxide (DMSO) at different concentrations, ranging from 5% to 20% supplemented with fetal calf serum (FCS), ranging from 0% to 90%. The functions of DMSO in the cryopreservation solution are to protect the cells from excessive dehydration during the freezing process and also to inhibit intracellular ice formation. Presumably, FCS protects the cells from injury induced by the generation of free oxygen radicals during the freeze cycle. The mixture of 10 % DMSO and 10% FCS in the cryopreservation solution can be regarded as optimal concentrations for these reagents in the freezing of liver cells.
Furthermore, it has been reported that different saccharides are able to enhance post-thaw cell viability by stabilizing cell membranes and proteins during the freezing procedures. Our attention was attracted by the disaccharide trehalose. Lower organisms e.g. fungi, yeasts, bacteria and insects have the ability to survive complete freezing and/or drying. All these organisms accumulate a large amount of disaccharides, especially trehalose, which is not found in the human body. Trehalose is a naturally-occurring disaccharide containing two glucose molecules bound in an alpha, alpha-1,1 linkage. We presumed that trehalose, when added to the standard cryopreservation solution, might protect human liver cells during freeze-thaw procedures. Our experiments were designed to compare cryopreservation solution (10% DMSO and 10% FCS, diluted in culture medium) with or without trehalose supplemtenation.

Figure 1: Primary human hepatocytes were isolated by using a modified two-step collagenase perfusion technique. Briefly, in step one, the specimens were flushed under sterile conditions with 500 ml of a pre-warmed (37oC) washing buffer containing 2.5 mM EGTA. During step two, the liver samples were digested by recirculation of 100 ml pre-warmed (37oC) digestion buffer with 0.05 % collagenase P (Roche, Germany). The tissue was then mechanically disrupted in washing buffer supplemented with 5% human albumin. The resulting cell suspension was filtered through a gauze-lined funnel, centrifuged (50 g, 5 min., 4oC) and resuspended in cold William`s medium E.

In order to find the optimal cell-protective dose, the effect of the trehalose concentration on the outcome of cryopreservation was evaluated in a pilot study: cells from three donors were frozen using the standard cryopreservation solution supplemented with 0.05, 0.10, 0.15, 0.20, 0.25 or 0.30 M trehalose. The concentration of trehalose with the best overall outcome was chosen for further experiments.During pilot experiments, the effect of various trehalose concentrations was investigated by determining the cell viability immediately after thawing, and the plating efficiency on day one of the culture. Additionally, a cell proliferation assay was performed on the third day of culture. Total protein, transaminase release and urea synthesis of the cells were also measured on culture day three. Thereafter, the comparison of standard cryopreservation solution (10% DMSO) with freezing medium containing trehalose in a concentration, previously found to be optimal, was performed (n = 9 donors) in order to analyze the post-thaw function of the cells during a culture period of seven days.
Besides the assessment of viability after thawing and plating efficiency of the cells on day one, a cell proliferation assay, as well as the measurement of total protein, release of transaminases, albumin and urea synthesis were performed on culture days 1, 2, 3, 5 and 7. Specific phase I and II enzyme activities of the liver cells were determined on the fifth day of culture.

The pilot study revealed that the post-thaw viability of liver cells frozen with a trehalose concentration of 0.1 to 0.3 M was increased compared to the control group. Plating efficiency was significantly higher for trehalose values of 0.15 and 0.2 M. The total protein and the cell proliferation assays showed optimal results with 0.2 and 0.3 M trehalose. Dynamics of enzyme levels in terms of AST and LDH leakage also seemed to be slightly lower for trehalose values of 0.2 to 0.3 M. No differences were observed in urea formation. Since the best overall outcome in most of the investigated parameters was observed with 0.2 M trehalose, this concentration was chosen for subsequent cryopreservation experiments.

Viability and plating efficiency – study with 0.2 M trehalose
Cell viability and plating efficiency of the freshly isolated primary human hepatocytes were 83.8 ± 6.1% and 77.2 ± 24.6%, respectively. After cryopreservation and subsequent thawing, these parameters were decreased in both experimental groups, when compared to initial levels. However, the post-thaw viability in the case of cryopreservation with trehalose was significantly higher than in the control group (62.9 ± 13 vs. 46.9 ± 11%; p < 0.01).
Attachment of cells to the collagen-coated plates was also improved significantly, compared to the control group, when 0.2 M trehalose was added to the cryopreservation solution (41.5 ± 18 vs. 17.6 ± 13%, p < 0.01).
Since the calculation of the plating efficiency based on the trypan blue exclusion test is not very precise, additional tests to assess our results were applied, including measuring total protein of the attached cells and a cell proliferation assay.

Total protein and cell proliferation assay
When trehalose was added to the cryopreservation solution, the total protein increased significantly in all culture days (Figure 2). Throughout the entire culture period, the bioreduction of the tetrazolium compound was observed higher in liver cells cryopreserved with trehalose (Figure 2). However, the difference between the two groups regarding the proliferation assay was not significant. The results of the protein measurement and the cell proliferation assay correlated with our findings concerning the viability and plating efficiency.

The morphology of the thawed hepatocytes attached to the collagen-coated plates was assessed throughout the culture period using light microscopy. The appearance of the cells was comparable with the morphology of the freshly isolated hepatocytes. They had the typical polygonal shape, granular cytoplasm with vesicular inclusions and one or multiple nuclei (Figure 3). Noticeable differences in morphological appearance between the two experimental groups were not observed. However, in most cases, we could confirm by light microscopy that the trehalose-treated group had more cells attached to the collagen-coated plates than the control group.

Enzyme release
The concentration of LDH and AST in the culture supernatants was measured to detect possible membrane leakage, indicating cell damage. In both groups, the highest level of LDH was recorded on the first day of culture, followed by a small decrease on day two. Throughout the rest of the culture period, LDH release was constant and remained at a low level. No significant differences between the two groups were detected. The peak of the AST release in both groups was also observed on the first day of culture. However, the AST concentration was significantly higher when no trehalose was added to the cryopreservation solution (613 ± 146 vs. 465 ± 103 U/day/106 plated cells, p < 0.001). A rapid decrease of the AST levels in both groups was observed on days two and three, followed by an almost stable course until the end of the culture period. However, the amount of AST on days three and seven was significantly higher in the group with trehalose compared to the control group.

Albumin and urea synthesis
The highest level of albumin secretion in both groups was observed 24 hours after plating. The initially high albumin value was larger in the trehalose group when compared to the control. However, the difference between the two groups was not statistically significant (Figure 4). In both groups, a decrease in the albumin synthesis rate was recorded after day one. During the entire culture period, albumin secretion was significantly higher for hepatocytes cryopreserved with a combination of trehalose and DMSO in the freezing solution.
Urea synthesis of the cryopreserved human hepatocytes slightly decreased from day one up until the end of the culture period. Although a higher level of urea secretion from the liver cells frozen with trehalose was observed for the first five days of culture, no significant differences between the two groups were found (Figure 4).

Phase I and II enzyme activities
Basal deethylation of the substrate 7-ehtoxycoumarin to the fluorescent product 7-hydroxycoumarin in human hepatocytes was detected only at a low level in both groups. The treatment of the cultures with 2 mM phenobarbital over a three day periods did not significantly stimulate the ECOD activity. No differences between the two groups in terms of catalyzation of 7-ethoxycoumarin were observed. Basal EROD activity was also low in both groups. However, after three days of treatment with 2.5 µM 3-MC, the EROD activity increased 25 fold in the cells cryopreserved with the addition of trehalose, whereas only 17 fold in the control group. This difference was not statistically significant (p > 0.05). Evaluation of the phase II enzyme activity showed no glucuronidation of the 4-MU in both experimental groups.