Liver Cell Transplantation: Basic Investigations for ... - SAGE Journals

0963-6897/09 $90.00 + .00 DOI: 10.3727/096368909X470775 E-ISSN 1555-3892 www.cognizantcommunication.com

Cell Transplantation, Vol. 18, pp. 777–786, 2009 Printed in the USA. All rights reserved. Copyright  2009 Cognizant Comm. Corp.

Liver Cell Transplantation: Basic Investigations for Safe Application in Infants and Small Children Jochen Meyburg,* Krassimira Alexandrova,† Marc Barthold,† Sabine Kafert-Kasting,† Andrea S. Schneider,‡ Masoumeh Attaran,‡ Friederike Hoerster,* Jan Schmidt,§ Georg F. Hoffmann,* and Michael Ott‡ *Department of General Pediatrics, University Children’s Hospital, 69120 Heidelberg, Germany †Cytonet GmbH & Co. KG, 30625 Hannover, Germany ‡Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, 30623 Hannover, Germany §Department of General and Visceral and Transplantation Surgery, University Hospital, 69120 Heidelberg, Germany Liver cell transplantation (LCT) is a very promising method for the use in pediatric patients. It is significantly less invasive than whole organ transplantation, but has the potential to cure or at least to substantially improve severe disorders like inborn errors of metabolism or acute liver failure. Prior to a widespread use of the technique in children, some important issues regarding safety and efficacy must be addressed. We developed a mathematical model to estimate total hepatocyte counts in relation to bodyweight to make possible more appropriate dose calculations. Different liver cell suspensions were studied at different flow rates and different catheter sizes to determine mechanical damage of cells by shear forces. At moderate flow rates, no significant loss of viability was observed even at a catheter diameter of 4.2F. Addition of heparin to the cell suspension is favored, which is in contrast to previous animal experiments. Mitochondrial function of the hepatocytes was determined with the WST-1 assay and was not substantially altered by cryopreservation. We conclude that especially with the use of small catheters, human LCT should be safe and efficient even in small infants and neonates. Key words: Hepatocyte transplantation; Cell application; Cryopreservation; Portal vein catheter; Children

INTRODUCTION During the last 10 years, liver cell transplantation (LCT) has become a promising alternative to whole organ transplantation in a variety of diseases. More than 100 patients have been treated so far, the majority suffering from acute liver failure or liver-based metabolic disorders (8). In pediatric patients, especially in infants and neonates, many technical issues have to be addressed due to anatomic limitations given by the small size of the patients. However, precise information in the literature on these technical issues is scarce. For example, the total number of liver cells in the adult liver is given in a frequently cited case report (9). Although the references provided there do not give appropriate information on the total hepatocyte count, dose calculations for applications in children have been based on that figure ever since. Because of the lack of fundamental technical information, we carried out basic investigations regarding appropriate doses, catheter size, flow velocities, aggregation of transplanted cells, effects of cryopreser-

vation, and effects of a cold liver cell suspension in vivo before the first use of LCT in our center. MATERIALS AND METHODS Total Hepatocyte Count Calculation of the total number of hepatocytes related to bodyweight was based on published findings. The proportional volume of hepatocytes is known to be 78– 80% of the total liver volume (14,20). Exact data on the volume of single human hepatocytes is scarce. For our calculation, we used findings from a histopathological study in 19 human liver grafts after reperfusion that measured a mean volume of 3734 ± 1056 µm3 (29). For determination of liver volume, we averaged the findings of four studies on liver volumetry by computed tomography (11,19,27,31), based on growth charts provided by the U.S. National Center for Health Statistics (http:// www.cdc.gov/nchs). The total hepatocyte number was then calculated from liver volume, proportional volume of hepatocytes, and single hepatocyte volume.

Received July 16, 2008; final acceptance January 2, 2009. Address correspondence to Jochen Meyburg, University Children’s Hospital, Im Neuenheimer Feld 150, D-69120 Heidelberg, Germany. Tel: +49 6221 5638428; Fax: +49 6221 565626; E-mail: [email protected]

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Isolation of Human Liver Cells Human liver cells were isolated from human liver tissue as described elsewhere (1). Briefly, the liver tissue was perfused with three different buffers, the last containing collagenase for the enzymatic digestion of the connective tissue. Mechanical disruption of the tissue releases the liver cells from the extracellular matrix. The harvested cell suspension was filtrated, washed, and resuspended in a physiological buffer. After addition of the cryopreserving solution containing dimethyl sulfoxide, the cells were controlled frozen and stored at below −140°C. Catheter Size and Flow Velocities For determination of the minimal tolerable catheter diameter, single lumen Broviac catheters with diameters of 4.2, 5.0, and 6.6 French were investigated (Vygon Lifecath Broviac, Vygon GmbH & Co. KG, Aachen, Germany). Specimens of three batches of cryopreserved human liver cells were used for the study. The cells differed in the viability after thawing (89%, 71%, and 37%); the mean cell concentration was 8.1 ± 0.7 × 106/ ml. The cell suspensions were quickly thawed in a water bath at 40°C, manually aspirated in a 50-ml syringe, and perfused at room temperature through the catheters at constant rates of 1, 2, 5, and 10 ml/h using a calibrated syringe pump (Asena CC MK III, Alaris Medical Systems, Baesweiler, Germany). The cell viability was determined by the trypan blue exclusion assay before and after the perfusion. Triplicates of each sample were measured, and the average values were used for analysis. Aggregate Formation Specimens from three batches of cryopreserved human liver cells (viability after thawing 45–79%) were investigated. After thawing, cells were centrifuged at 50 × g, 5 min, 4°C and incubated in a buffer containing 0, 1, 2, 5, or 10 U heparin/ml. Freshly thawed cells without addition of heparin served as additional control. After an incubation period of 20–60 min, samples of each assay were investigated in a counting chamber (Neubauer improved). Cell aggregates of more than four cells were registered. Effects of Cryopreservation Effects of cryopreservation were studied in samples of two batches of cryopreserved human liver cells: HLK023 (donor age 9 days, viability after thawing 76 ± 2%) and HLK020 (donor age 41 years, viability after thawing 78 ± 2%). These batches were compared with cryopreserved cells of an immortalized hepatoma cell line (HEP-G2; ATCC) and cryopreserved liver cells (Res053) isolated from discarded tissue after human liver resection (resection specimen) that also served as a

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control sample of each test run. In a first series, different cell concentrations were tested to prove the specificity of the test and to find out the most suitable cell quantity. Samples of the same cell batches were then tested immediately after thawing, after 24 and 48 h. In addition to these investigations on cryopreserved cells, fresh liver cells from three resection specimens (Res051 with viability fresh/cryo 83%/74%; Res052 with viability 66%/ 51%; Res054 with viability 87%/68%; and Liv046 with viability 75/65%) were studied before and after cryopreservation. To investigate mitochondrial function of the hepatocytes, the formation of formazan from the tetrazolium salt WST-1 (Cell Proliferation Reagent WST-1, Roche Diagnostics, Mannheim) by the mitochondrial succinate dehydrogenase system was measured. Liver cells were seeded on 24-well collagen I-coated plates after isolation or after thawing, respectively. Different concentrations of 0.065, 0.125, 0.25, and 0.5 × 106 viable cells per well were used in the first experiment; 0.5 × 106 viable cells per well was chosen as standard seeding concentration thereafter. Depending on the viability the total cell count ranged between 0.6 × 106 and 0.98 × 106 per well. The volume was 1 ml/well in all cases. Cells were incubated in Cytonet cell culture medium A (Cytonet Hannover GmbH & Co. KG), containing insulin, dexamethasone, and FCS, without growth factors. No medium changes were performed after the cells were plated and during the entire test phase. The cells were partially attached on the collagen layer on the plate bottom and partially in suspension. After 30-min incubation in the medium at 37°C, 5% CO2 and 95% humidity WST-1 reagent were added and incubated for 1 h. The absorbance of the metabolized product at 450 nm was measured by spectrophotometer Tecan Spectrafluor Plus. The validity of every rest run was proven by simultaneously testing of one vial of the control liver cells (batch Res035). Depending on availability the tests of the cryopreserved cells were repeated two or three times. The fresh cells were tested in single assays. Triplicates of each sample were measured. For analysis, were used the average values. RESULTS Total Hepatocyte Count Similar to other childhood growth curves, the calculated total hepatocyte counts related to age followed a sigmoid curve rather than a straight line (Fig. 1A). The most commonly used dose of 0.2 × 109 hepatocytes/kg of bodyweight is derived from an assumed total numer of 280 × 109 hepatocytes in the liver of an adult weighing 70 kg. According to this calculation, the hepatocyte count per kilogram bodyweight would be 4 × 109, and 0.2 × 109 would represent 5% of that number (9). Our

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Figure 1. Age-related changes in the calculated total hepatocyte count, expressed as mean (solid line) and standard deviation (dotted lines). (A) Ccalculation based on mean values for liver and hepatocyte volumes. (B) Calculation based on mean and standard deviations of liver and hepatocyte volumes.

calculations of 5% hepatocytes/kg differed from that commonly used figure. Due to the nonlinear increase in total liver cell count (Fig. 1A), higher numbers were calculated, especially in infants and small children (Fig. 2A). Our calculations were based on mean values for hepatocyte and liver volumes. However, large variations in these measurements have to be taken into account. Thus, our observations were clearly less evident when standard deviations for liver and hepatocyte volumes were considered (Figs. 1B and 2B).

Catheter Size and Flow Velocities The passage of liver cells through the three narrow catheters always caused a detectable loss of viability (Fig. 3). However, the loss of viability was clearly dependent on the cell quality, on the catheter size, and on the applied flow rate. Cells with excellent viability rates (89%) showed only marginal changes in viability independent of catheter size or flow rate (Fig. 3, upper panel). The changes were more pronounced in the medium via-

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Figure 2. Commonly used doses in human liver cell applications. The dashed line indicates the most frequently used dose of 0.2 × 109 hepatocytes/kg. The curved lines represent 5% of the calculated liver cell count per kilogram, expressed as mean (soild line) and standard deviation (dotted lines). (A) Calculation based on mean values for liver and hepatocyte volumes. (B) Calculation based on mean and standard deviations of liver and hepatocyte volumes.

bility cells (71%), but still no major differences were observed with regards to catheter size or flow rates (Fig. 3, middle panel). Cells of rather poor viability (37%) get easily damaged at higher flow rates above 2 ml/min (Fig. 3, lower panel). These changes were similar between the three catheter sizes. Cell Aggregate Formation The cell aggregation in relation to heparin content of the cell suspension is summarized in Table 1. No signifi-

cant differences were found between the three individual cell batches. Heparin concentrations from 1 to 10 U/ml cell suspension did not cause significant formation of cell aggregates in contrast to findings in the animal model (18). Effects of Cryopreservation The results of the WST-1 testing are given in Figure 4. In the validation experiment, we measured an increase of WST-1 absorption that was proportional to the cell


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Figure 3. Catheter sizes and flow velocities. Vertical bars show the loss of viability caused by the passage of liver cells through three catheters (6.6F, 5F, and 4.2F). Results are shown for three batches of cells with excellent (upper panel), mean (middle panel), and rather poor (lower panel) viability rates after thawing.

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Table 1. Aggregate Formation of Three Different Liver Cell Batches in Relation to Heparin Content of the Suspension Batch No. 1 Control Washed cells w/o heparin 1 U/ml heparin 2 U/ml heparin 5 U/ml heparin 10 U/ml heparin

2

3

Mean

% Aggregated Cells

1.5

0

4

1.8 ± 2.1

6.1 ± 7.1

2.5 0.5 0.5 0.5 1

0 0.5 1 0 0

3.5 3.5 6 2 3

2.0 ± 1.9 1.5 ± 1.9 2.5 ± 2.9 0.8 ± 1.0 0.8 ± 1.5

6.7 ± 6.3 5.0 ± 6.2 8.3 ± 9.6 2.8 ± 3.3 2.8 ± 5.0

n = 2 independent counts per value.

concentration in all four cell batches (Fig. 4A). The highest cell concentration (0.5 × 106 viable cells/well) was used for all experiments thereafter. All studied cryopreserved cells had good WST-1 values after thawing, indicating intact mitochondrial function (Fig. 4B). In contrast to the immortalized HEP-G2 cells, which showed similar high absorbance rates 24 and 48 h after thawing, the values of the Res035 hepatocytes markedly decreased after 24 and 48 h. Similar findings were observed in the neonatal liver cells (HLK023): very high metabolic activity immediately after thawing followed by a drastic decrease of this parameter during the next 24 to 48 h of cultivation. The initial absorbance values for the adult cryopreserved liver cells (HLK020) were about 50% lower, but the decline during the next 2 days was considerably slower. The direct study of the viability of fresh versus the respective cryopreserved cells showed controversial results (Fig. 4C). In two of the four tested batches (Res051 and Res052) the metabolic activity decreased by about 40% after cryopreservation. In the other two batches, the measured activity after cryopreservation was surprisingly higher than that of the corresponding fresh cells. DISCUSSION In 1998, the first long-term success of LCT in a 10year-old girl with Crigler-Najjar syndrome type I prompted further clinical trials in children with inherited metabolic diseases (9). For this case, dose calculations were based on a total number of hepatocytes of 280 × 109 in the adult liver. Given an average body weight of 70 kg for an adult, the total hepatocyte count in pediatric patients was assumed to be 4 × 109 cells/kg. According to animal experiments, the transfer of 5% of enzyme activity was regarded appropriate, which corresponds to 0.2 × 109 cells/kg. This calculation has been referred to ever since for pediatric trials of LCT. However, it is unlikely that the number of liver cells should increase

linearly in contrast to all other sigmoid growth curves in infancy and childhood. Our calculation of hepatocyte count per milliliter liver tissue from morphometric data corresponds well to previous findings (14), and related to age-dependent liver volume, the resulting curve is sigmoid as expected. However, our model calculation has two major limitations. First, large variations of liver and hepatocyte volume do not allow exact prediction of liver cell counts. Second, our data are based on the assumption that the increase in liver volume throughout childhood is mainly the result of an increase in hepatocyte number without significant changes in hepatocyte size. Although individual studies suggest some developmental changes of hepatocyte volume in rats (21) and mice (13), no human data are available in the literature. We also investigated different cell suspensions without finding significant differences in hepatocyte size between adult and pediatric donors. Considering these limitations, our calculations should not be regarded as exact estimations of total liver cell count, but rather as a model for basic considerations about safety and efficacy of LCT in children. Our data suggest that the commonly used cell dose derived from the original calculation by Fox et al. (9) is probably less than 5% of the total liver cell count, and that differences are more pronounced in infants and young children (Fig. 2). Because this age group is most relevant for pediatric LCT, our model has important safety implications. Severe side effects of LCT have not been reported in children so far, but it is well known from animal studies that the most severe complications of LCT, portal vein thrombosis and pulmonary embolism, are dose dependent. We used a modification of our model to calculate weight-related total hepatocyte counts in rats, mice, rabbits, and pigs and found that severe side effects in animal studies did only occur at cumulative doses much higher than 5%. Therefore, the commonly used dose of 0.2 × 109 cells/kg, which seems to be considerably less than 5% of total liver cell count in small children, should be safe and might even be increased without substantial risk of severe side effects. The exact cell dose that is needed for phenotypic correction of hepatic-based metabolic diseases can only be estimated. The assumption that 5% will correct CriglerNajjar syndrome (9) was derived from animal data in Gunn rats. For other inborn errors of metabolism, such data are missing. In urea cycle disorders, enzyme activities of healthy heterozygotes are usually above 25% (15), but living-related liver transplantations from heterozygous parents have shown that as little as 8% of enzyme activity may be sufficient to achieve phenotypic correction (5). It is important to keep in mind that only about 30% of the transplanted hepatocytes engraft permanently in animal experiments (10). If this is also the case


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Figure 4. Measured absorbance of the WST-1 reagent at 450 nm. (A) Absorbances at different cell concentrations. (B) Changes over 48 h in cryopreserved cells. (C) Direct effects of cryopreservation in fresh cells from liver resectates.

in human LCT, transplatation of 5% hepatocytes would result in only about 1.5% of permanently engrafting cells. Looking at our calculations, these figures seem to be even lower, especially in young children. However, substantial metabolic improvement after LCT has been described in most pediatric patients so far in spite of using doses far too low to provide 5–10% cell engraftment (16,23–25). In one patient, up to 3.8% of enzyme activity could be measured in a liver biopsy 8 months

after transplantation of 0.2 × 109 liver cells/kg, corresponding to 1.5% permanently engrafting hepatocytes (24). Therefore, issues like proliferation of the engrafted cells, upregulation of specific enzyme activities, quality of the donor cells, and multiple courses of cell transfer seem to be much more important for LCT efficacy than exact dose calculations. Among the children suitable for LCT are many that could benefit from the technique early in infancy or even

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during the neonatal period. In these patients, the small size of the portal vein and its tributaries limits the size of the application catheter. It seems likely that, at least at higher flow rates, the cells may be damaged by shear forces occurring in small catheters as has been shown for neonatal ECMO (28). From the published cases of pediatric LCT it cannot be concluded what catheter size is sufficient to avoid damage of the transfused cells. Sizes of 5F (2), 6F (23), and 7F (7,12) have been reported for either surgically introduced or radiologically placed (transhepatic or via the umbilical vein) portal vein catheters, but in the majority of cases, catheter sizes are not given. We decided to investigate the effect of shear forces in Hickman catheters of different diameters. Such catheters allow fractionated and repeated applications of liver cells. These strategies are thought to minimize the risk of portal vein thrombosis and extrahepatic shunting into the pulmonary circulation. Our findings show that even the smallest 4.2F catheter was sufficient to deliver cells at adequate flow rates with sufficient residual viability. Only hepatocytes of considerably low viability after thawing are at risk for substantial damage when infused through the smallest catheter, which can still be avoided by using low flow rates. We did not observe a significant loss of viability of the cryopreserved cells within the first 60 min after thawing (data not shown). A typical pediatric application protocol would use a total cell dose of 0.2 × 109 vital cells/kg bodyweight given in six individual applications. Given a mean cell concentration of 0.8 × 106 cells/ml and a mean vitality of 75%, this would mean a volume of 5.5 ml/kg for the individual application. To keep application time below 60 min, application rates of 1 ml/min may be used for a bodyweight up to 10 kg, and 2 ml/kg for older children up to 20 kg. Thus, a 4.2F Hickman catheter seems most appropriate for LCT in neonates, infants, and small children. Larger catheters and flow rates of 5 ml/min can be used in older children without the risk of cell damage by shear forces. Under clinical conditions, relevant thromboses related to LCT have been reported in two adults so far. One of these patients received liver cells for the treatment of graft failure following liver transplantation despite a preexisting stenosis of the portal venous anastomosis (4). Another patient who apparently experienced a thrombosis of the mesenterical vein following LCT for acute liver failure is mentioned in a recent review of clinical application of LCT, but no details are given on that case (8). A reduction of the portal vein flow and/or an increase in portal venous pressure has been shown in several animal studies of LCT (3,18,22,30) and may favor the formation of portal vein thrombosis also in human LCT. Furthermore, the cell suspension itself may exert procoagulatory effects. Tissue factor-related acti-

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vation of the coagulation cascade was demonstrated recently in cryopreserved human hepatocytes (26), confirming previous observations in isolated pancreatic islet cells (17). Beyond macroscopic portal vein thrombosis, such activation of the coagulation cascade is associated with lower engraftment rates in islet cell transplantation, and it is likely that this is also relevant for human LCT. Tissue factor can be effectively inhibited by antithrombin in combination with heparin. Paradoxically, heparin was found to increase clot formation in a pig model of LCT for acute liver failure (18). Our results show that in vitro heparin in various doses does not cause significant hepatocyte aggregation. Therefore, we suggest to add heparin to the hepatocyte suspension at concentrations of 2–5 U/ml, which are commonly used to keep arterial or central venous lines open. One of the main advantages of LCT for human application is the possibility to cryopreserve and store isolated hepatocytes for elective use. In most clinical trials of LCT, a combination of fresh and cryopreserved cells has been transplanted. The first patient in which only cryopreserved liver cells were used successfully was a 14-month-old boy with ornithine transcarbamylase deficiency (25). This urea cycle defect is caused by a defective mitochondrial enzyme, thus intact mitochondria after thawing of the cryopreserved liver cells are mandatory. Regarding typical indications for human LCT, intact mitochondria are necessary not only for the treatment of urea cycle disorders, but for a variety of inborn errors of metabolism and also acute liver failure. The WST-1 assay represents a reliable and quite easy to use method for the determination of mitochondrial function (6). In principle, it measures the mitochondrial enzyme succinate dehydrogenase, which is involved in the production of ATP. We demonstrated excellent viability values after thawing for neonatal liver cells, which did not significantly differ from freshly isolated or immortalized Hep-G2 cells. However, after 24-h cultivation a distinct reduction of mitochondrial function was observed. In the adult cells, the initial WST-1-absorbances were lower, but the decline after 24 and 48 h was less pronounced. Because it is well known from animal experiments that engraftment of transplanted hepatocytes is completed after 16–20 h (10), a decline in mitochondrial function in vivo after 24 h does not seem to be relevant for the clinical application. We expected to find a decrease of the WST-1 absorptions in the four batches of fresh cells that were investigated before and after cryopreservation. Surprisingly, this was only found in two of the four batches, while the values even increased after cryopreservation in the other two. What cellular mechanisms might have increased the ability to metabolize formazan remained unclear. We conclude from our findings that mitochondrial function is preserved to a large


extent after cryopreservation in the studied liver cells. Whether the higher values of mitochondrial function testing in the neonatal cells imply a better function or a survival advantage in vivo remains to be investigated. In conclusion, we give answers to important practical questions that are relevant for the clinical use of LCT, especially in small children and infants. According to our mathematical model and in contrast to published data, the commonly used dose of 0.2 × 109 seems safe and might even be increased in infants and young children without substantial risk for plugging or shunting of the cells. However, issues other than exact dose calculations are important for the efficacy of LCT. Catheters as small as 4.2F do not damage transfused liver cells by shear forces, and their use in combination with small doses of heparin should minimize and not increase the risk of portal vein thrombosis and failure to engraft. Because cryopreservation apparently does not significantly alter the mitochondrial function of the liver cells, cryobanking of the cells and elective use in a variety of patients and conditions appears possible. REFERENCES 1. Alexandrova, K.; Griesel, C.; Barthold, M.; Heuft, H. G.; Ott, M.; Winkler, M.; Schrem, H.; Manns, M. P.; Bredehorn, T.; Net, M.; Manyalich I Vidal, M.; Kafert-Kasting, S.; Arseniev, L. Large-scale isolation of human hepatocytes for therapeutic application. Cell Transplant. 14(10): 845–853; 2005. 2. Allen, K. J.; Cheah, D. M.; Wright, P.; Brooks, M.; Angus, P.; Jones, R.; Williamson, R.; Hardikar, W. Immunological considerations in liver cell transplantation for Crigler-Najjar syndrome. J. Gastroenterol. Hepatol. 20(Suppl.):A71; 2005. 3. Attaran, M.; Schneider, A.; Grote, C.; Zwiens, C.; Flemming, P.; Gratz, K. F.; Jochheim, A.; Bahr, M. J.; Manns, M. P.; Ott, M. Regional and transient ischemia/reperfusion injury in the liver improves therapeutic efficacy of allogeneic intraportal hepatocyte transplantation in lowdensity lipoprotein receptor deficient Watanabe rabbits. J. Hepatol. 41(5):837–844; 2004. 4. Baccarani, U.; Adani, G. L.; Sanna, A.; Avellini, C.; Sainz Barriga, M.; Lorenzin, D.; Montanaro, D.; Gasparini, D.; Risaliti, A.; Donini, A.; Bresadola, F. Portal vein thrombosis after intraportal hepatocytes transplantation in a liver transplant recipient. Transpl. Int. 18(6):750–754; 2005. 5. Ban, K.; Sugiyama, N.; Sugiyama, K.; Wada, Y.; Suzuki, T.; Hashimoto, T.; Kobayashi, K. A pediatric patient with classical citrullinemia who underwent living-related partial liver transplantation. Transplantation 71(10):1495–1497; 2001. 6. Cook, J. A.; Mitchell, J. B. Viability measurements in mammalian cell systems. Anal. Biochem. 179(1):1–7; 1989. 7. Darwish, A. A.; Sokal, E.; Stephenne, X.; Najimi, M.; de Goyer Jde, V.; Reding, R. Permanent access to the portal system for cellular transplantation using an implantable port device. Liver Transpl. 10(9):1213–1215; 2004. 8. Fisher, R. A.; Strom, S. C. Human hepatocyte transplanta-

785

9.

10.

11. 12.

13.

14.

15.

16.

17.

18.

19. 20.

21.

22.

tion: worldwide results. Transplantation 82(4):441–449; 2006. Fox, I. J.; Chowdhury, J. R.; Kaufman, S. S.; Goertzen, T. C.; Chowdhury, N. R.; Warkentin, P. I.; Dorko, K.; Sauter, B. V.; Strom, S. C. Treatment of the Crigler-Najjar syndrome type I with hepatocyte transplantation. N. Engl. J. Med. 338(20):1422–1426; 1998. Gupta, S.; Rajvanshi, P.; Sokhi, R.; Slehria, S.; Yam, A.; Kerr, A.; Novikoff, P. M. Entry and integration of transplanted hepatocytes in rat liver plates occur by disruption of hepatic sinusoidal endothelium. Hepatology 29(2):509– 519; 1999. Heinemann, A.; Wischhusen, F.; Puschel, K.; Rogiers, X. Standard liver volume in the Caucasian population. Liver Transpl. Surg. 5(5):366–368; 1999. Horslen, S. P.; McCowan, T. C.; Goertzen, T. C.; Warkentin, P. I.; Cai, H. B.; Strom, S. C.; Fox, I. J. Isolated hepatocyte transplantation in an infant with a severe urea cycle disorder. Pediatrics 111(6 Pt. 1):1262–1267; 2003. Kanai, K.; Kanamura, S.; Watanabe, J. Peri- and postnatal development of heterogeneity in the amounts of endoplasmic reticulum in mouse hepatocytes. Am. J. Anat. 175(4): 471–480; 1986. Krishnamurthy, G. T. Morphology and microstructure of the hepatobiliary system. In: Krishnamurthy, G. T.; Krishnamurthy, S., eds. Nuclear hepatology. Heidelberg, Germany: Springer; 2000:1–19. Lee, B.; Yu, H.; Jahoor, F.; O’Brien, W.; Beaudet, A. L.; Reeds, P. In vivo urea cycle flux distinguishes and correlates with phenotypic severity in disorders of the urea cycle. Proc. Natl. Acad. Sci. USA 97(14):8021–8026; 2000. Mitry, R. R.; Dhawan, A.; Hughes, R. D.; Bansal, S.; Lehec, S.; Terry, C.; Heaton, N. D.; Karani, J. B.; Mieli Vergani, G.; Rela, M. One liver, three recipients: Segment IV from split-liver procedures as a source of hepatocytes for cell transplantation. Transplantation 77(10):1614–1616; 2004. Moberg, L.; Johansson, H.; Lukinius, A.; Berne, C.; Foss, A.; Kallen, R.; Ostraat, O.; Salmela, K.; Tibell, A.; Tufveson, G.; Elgue, G.; Nilsson Ekdahl, K.; Korsgren, O.; Nilsson, B. Production of tissue factor by pancreatic islet cells as a trigger of detrimental thrombotic reactions in clinical islet transplantation. Lancet 360(9350):2039–2045; 2002. Muraca, M.; Neri, D.; Parenti, A.; Feltracco, P.; Granato, A.; Vilei, M. T.; Ferraresso, C.; Ballarin, R.; Zanusso, G. E.; Giron, G.; Rozga, J.; Gerunda, G. Intraportal hepatocyte transplantation in the pig: Hemodynamic and histopathological study. Transplantation 73(6):890–896; 2002. Noda, T.; Todani, T.; Watanabe, Y.; Yamamoto, S. Liver volume in children measured by computed tomography. Pediatr. Radiol. 27(3):250–252; 1997. Phillips, M. J.; Satir, P. The Hepatocyte: Organization. In: Arias, I. M.; Jakoby, W. B.; Popper, H.; Schachter, D.; Shafritz, D. A., eds. The liver. Biology and pathobiology. New York: Raven Press; 1988:9–10. Schmucker, D. L.; Mooney, J. S.; Jones, A. L. Stereological analysis of hepatic fine structure in the Fischer 344 rat. Influence of sublobular location and animal age. J. Cell Biol. 78(2):319–337; 1978. Schneider, A.; Attaran, M.; Gratz, K. F.; Bleck, J. S.; Winkler, M.; Manns, M. P.; Ott, M. Intraportal infusion of 99mtechnetium-macro-aggregrated albumin particles and hepatocytes in rabbits: assessment of shunting and portal hemodynamic changes. Transplantation 75(3):296–302; 2003.

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23. Sokal, E. M.; Smets, F.; Bourgois, A.; Van Maldergem, L.; Buts, J. P.; Reding, R.; Bernard Otte, J.; Evrard, V.; Latinne, D.; Vincent, M. F.; Moser, A.; Soriano, H. E. Hepatocyte transplantation in a 4-year-old girl with peroxisomal biogenesis disease: Technique, safety, and metabolic follow-up. Transplantation 76(4):735–738; 2003. 24. Stephenne, X.; Najimi, M.; Sibille, C.; Nassogne, M. C.; Smets, F.; Sokal, E. M. Sustained engraftment and tissue enzyme activity after liver cell transplantation for argininosuccinate lyase deficiency. Gastroenterology 130(4): 1317–1323; 2006. 25. Stephenne, X.; Najimi, M.; Smets, F.; Reding, R.; de Goyet, J. V.; Sokal, E. M. Cryopreserved liver cell transplantation controls ornithine transcarbamylase deficient patient while awaiting liver transplantation. Am. J. Transplant. 5(8):2058–2061; 2005. 26. Stephenne, X.; Vosters, O.; Najimi, M.; Beuneu, C.; Dung, K. N.; Wijns, W.; Goldman, M.; Sokal, E. M. Tissue factor-dependent procoagulant activity of isolated human hepatocytes: relevance to liver cell transplantation. Liver Transpl. 13(4):599–606; 2007. 27. Urata, K.; Kawasaki, S.; Matsunami, H.; Hashikura, Y.;

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28.

29.

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Ikegami, T.; Ishizone, S.; Momose, Y.; Komiyama, A.; Makuuchi, M. Calculation of child and adult standard liver volume for liver transplantation. Hepatology 21(5): 1317–1321; 1995. Van Meurs, K. P.; Mikesell, G. T.; Seale, W. R.; Short, B. L.; Rivera, O. Maximum blood flow rates for arterial cannulae used in neonatal ECMO. ASAIO Trans. 36(3): M679–681; 1990. Vertemati, M.; Sabatella, G.; Minola, E.; Gambacorta, M.; Goffredi, M.; Vizzotto, L. Morphometric analysis of primary graft non-function in liver transplantation. Histopathology 46(4):451–459; 2005. Wilhelm, A.; Leister, I.; Sabandal, P.; Krause, P.; Becker, H.; Markus, P. Acute impairment of hepatic microcirculation and recruitment of nonparenchymal cells by intrasplenic hepatocyte transplantation. J. Pediatr. Surg. 39(8): 1214–1219; 2004. Yu, H. C.; You, H.; Lee, H.; Jin, Z. W.; Moon, J. I.; Cho, B. H. Estimation of standard liver volume for liver transplantation in the Korean population. Liver Transpl. 10(6): 779–783; 2004.