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Original Contribution

REPEATED INTRAPORTAL HEPATOCYTE TRANSPLANTATION IN ANALBUMINEMIC RATS

JACEK ROZGA, 1 MICHAEL HOLZMAN, ALBERT D. MOSCIONI, HIKARO FUJIOKA, EUGENIO MORSIANI, AND ACHILLES A. DEMETRIOU

Department of Surgery and Liver Support Unit, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Suite 8215, Los Angeles, CA 90048

[ ] Abstract - The optimal site for implantation of isolated hepatocytes has not been established. We have developed a novel technique which allows repeated infusion of hepato- cytes into the portal system via an indwelling catheter. Seven Nagase Analbuminemic rats (NAR) underwent single intra- portal infusion of 2 × 10 7 isolated normal albumin-producing rat hepatocytes. Another seven NAR rats underwent place- ment of indwelling catheters into the portal venous system via the gastroduodenal vein. Each of them received six batches of 5 × 106 normal albumin producing hepatocytes. Seven control NAR rats were infused rel~eatedly (intrapor- tally) with saline only. Plasma albumin (ELISA) showed sig- nificant increase in experimental animals and was more pronounced (p < 0.05) in rats transplanted repeatedly than in those given a single dose of cells. Imraunohistochemical staining of the liver sections confirmed the presence of trans- planted albumin producing hepatocytes. Rats transplanted with a single large batch of isolated hepatocytes showed liver tissue damage, whereas those subjected to repeated cell infu- sions had normal liver histology. We have developed a novel intraportal transplantation method which allows successful engraftment of a large number of isolated hepatocytes.

[ ] Keywords - Hepatocyte; Liver; Transplantation; Rat.

INTRODUCTION

Although significant progress has been achieved in iso- lated hepatocyte transplantation, the optimal method has not yet been determined. Recently our laboratory has shown that the intrahepatic location may be an ex- cellent site for grafting (6). However, with single in- jection only a limited number of hepatocytes can be transplanted without causing portal hypertension and liver tissue necrosis (5).

In this study we describe a novel technique allowing for repeated intraportal infusions of isolated hepato-

cytes through an implantable portal vein access device. With this method, we were able to transplant a large number of cells without producing any damage to the liver parenchyma. Using Nagase analbuminemic rat (NAR) model, we have also demonstrated a significant and prolonged functional effect of transplanted nor- mal rat hepatocytes.

MATERIALS AND METHODS

Animals Male Sprague-Dawley rats (250-300 g) were pur-

chased from Harlan Sprague-Dawley, Inc. (Indianap- olis, IN). Analbuminemic rats were obtained through the courtesy of Dr. S. Nagase (Sasaki Institute, Tokyo, Japan) and were maintained as congeneic strains by in- breeding and backcrossing at the local animal care fa- cility. Animals were fed standard laboratory rat chow (Rodent Chow 5001, Ralston Purina, St. Louis, MO) and given tap water ad lib. Animals were housed in cli- mate controlled rooms at 21°C and 12 h light/dark cycle.

Chemicals Collagenase (type IV ), purified rat albumin, bovine

serum albumin and other chemicals used for liver per- fusion and ELISA assays were purchased from Sigma Chemical Co. (St. Louis, MO). Purified rabbit IgG anti-rat albumin and rabbit anti-rat albumin peroxi- dase conjugate were purchased from Research Plus Co. (Bayonne, N J). Tissue culture media were pur- chased from Sigma Chemical Co. (St. Louis, MO). Cy- closporine (Sandimmune) was purchased from Sandoz

Accepted 8/31/94.

237

lTo whom correspondence should be addressed.

238 Cell Transplantation • Volume 4, Number 2, 1995

doz (East Hanover, N J). Vectastain ABC kits (Rabbit IgG) were purchased from Vector Laboratories, Inc. (Burlingame, CA).

infused over a 2-min period. The catheter and the chamber were subsequently flushed with 2 mL of hep- arinized saline (Fig. 2).

Preparation of Hepatocytes Donor rat hepatocytes were harvested using the por-

tal vein EDTA/collagenase digestion as earlier de- scribed (6). The parenchymal cells were washed three times using Dulbecco's modified Eagle's medium and passed through a 50 tz mesh to arrive at truly isolated hepatocytes. Hepatocytes were further purified by sed- imentation on Percoll density gradient (8). Viability was determined by trypan blue exclusion and the final cell suspension always contained over 95% viable hepatocytes.

Implantation of Indwelling Vascular Catheter Rats underwent a midline laparotomy under ether

anesthesia. A small SC pocket in the left groin was cre- ated for placement of the vascular access device (Mini- Port; Pharmacia Deltec Inc., St. Paul, MN)which was filled with heparinized saline (I0 U/mL) (Fig. 1). The port was connected to a silicone tubing (Medical-Grade Tubing, Dow Corning, O.D. 0.625 ram) which was tun- neled subcutaneously and brought into the peritoneal cavity through a small puncture in the anterior abdom- inal wall. It was then placed in the gastroduodenal vein at the junction of the portal vein.

Infusion of Hepatocytes During each cell infusion, the Mini-Port chamber

was punctured percutaneously and 5 × 106 hepato- cytes suspended in 3 - 4 mL of heparinized saline were

Experimental Design

Group L Seven NAR rats were given intraportally a single dose of 2 x 10 v isolated Sprague-Dawley rat hepatocytes. The single-cell suspension was selectively infused into the anterior liver lobes (70% liver mass) to ensure early portal decompression through the non- transplanted liver lobes. This technique is described in detail elsewhere (6).

Group II. Seven NAR rats underwent placement of indwelling vascular access devices and received infu- sions of 5 × 106 isolated Sprague-Dawley rat hepato- cytes on days 1, 3, 5, 8, 10, and 12.

Group IlL Seven NAR rats served as controls. They underwent placement of indwelling vascular access de- vices and received infusions of heparinized saline (4 mL) on days 1, 3, 5, 8, 10, and 12.

All animals received Cyclosporin A (10 mg/kg BW intramuscularly) one day prior to cell transplantation and then every other day until sacrifice.

Blood samples were obtained from the rat tail vein at days 0, 7, 14, 21, and 28 for determination of plasma albumin. All animals were killed at 28 days. At sacri- fice, liver and lung tissue specimens were fixed in 4070 buffered formalin.

Plasma Albumin Determination Quantitative analysis of albumin in plasma samples

was carried out by Sandwich ELISA, using a modi-

Fig. 1. Implantable vascular access device used in this study.

Hepatocyte t ransplantat ion • J.R. ROZGA ET A~. 239

Fig. 2. Implantable vascular access device was placed in the left groin. It was punctured percutaneously and isolated rat he- patocytes were infused over a period of 2 min. After injection, the chamber was filled with heparinized saline.

fled double antibody assay as earlier described (6). All samples and standards were run in duplicate. Plasma albumin levels were confirmed qualitatively by 10°70 SDS-PAGE (9); purified rat albumin and normal Sprague-Dawley rat plasma were used for reference. The specificity of the assay was confirmed by carrying out immunotransblots using rabbit anti-rat albumin antiserum (1).

Histology Light microscopy was carried out on liver and lung

sections stained with hematoxylin and eosin. In addition, albumin-positive cells were sought on gelatin-coated slides immunostained utilizing rabbit anti-rat albumin IgG antibodies and an immunoperoxidase avidin-bio- tin peroxidase complex (Vectastain ABC) method (7).

Statistical Analysis Statistical analysis of the data was carried out using

one-way ANOVA. The values are giw;n as mean + SD.

RESULTS

A significant (P < 0.01) and sustained increase in plasma albumin concentration occurred in both groups of NAR rats transplanted with normal albumin- producing Sprague-Dawley rat hepatocytes (Fig. 3). Group II NAR rats subjected to repeated cell infusions showed at 28 days higher albumin levels than Group

I NAR rats transplanted with a single dose of hepato- cytes (P < 0.05). Group III controls showed no change in plasma albumin concentration throughout the ex- perimental period. The ELISA findings were con- firmed by 10°70 SDS-PAGE. In both Group I and Group II NAR rats, the increase in protein band seen on the SDS-PAGE was shown to be albumin by pro- tein immunoblot, utilizing rabbit anti-rat albumin IgG. No significant changes in total plasma protein were found after transplantation with either single or re- peated doses of normal rat hepatocytes.

On light microscopy, Group I rats transplanted with a large single dose of isolated albumin-producing he- patocytes showed in some sections areas of tissue scar- ring. Fibrosis appeared around some of the portal and hepatic veins and large macrophages with brown lipo- fuscin pigment were occasionally seen between the liver cell plates. In contrast, liver sections from Group II rats subjected to intraportal infusion of six small batches of isolated hepatocytes showed no abnormal changes.

Immunohistochemically, liver sections from NAR rats infused with saline showed very few albumin- positive cells, mostly as singlets (Fig. 4). In contrast, Group I NAR rats transplanted with normal albumin- producing rat hepatocytes showed numerous albu- min-positive cells. They were uniformly distributed throughout the liver parenchyma and were sometimes arranged in small clusters containing 5-20 cells (Fig. 5). Liver sections obtained from Group II rats showed ex-

240 Cell Transplantation • Volume 4, Number 2, 1995

5 0 Albumin (m g/dl) 40 / ~ m u l t i p l e transpl.

/ p <0.05

30

20 I ~ s i n g l e transpl.

j ; / to I'

r ~ ? p controls 0 I o "Baseline 7 clays 14 'days'21 'days" 28 'days' Time

Fig. 3. Graph showing plasma albumin levels in the experimental animals at 0, 7, 14, 21, and 28 days posttransplantation. Control analbuminemic rats were given repeated injections of normal saline.

ceptionally large number of cells that stained positive for albumin (Fig. 6a). They were frequently seen as large clusters (Fig. 6b). Transplanted cells produced no changes in the hepatic micro-architecture. Engraftment

was so perfect that the transplanted cells could be iden- tified within the apparently normal lobules only immu- nohistochemically. No evidence of liver cell emboli in the lungs was found.

Fig. 4. Analbuminemic control rats injected with normal saline had not more than few albumin-positive cells per liver sec- tion. This is the usual background seen in the liver of NAR rats (Vectastain ABC method; magnif. 35 x).

Hepatocyte transplantation • J.R. ROZGA ET AL. 241

Fig. 5. Liver sections obtained from analbuminemic rats transplanted intraportally with a single does of 2 x 10 7 normal rat hepatocytes showed a moderate number of albumin-positive cells (1-20). They were randomly distributed throughout the liver parenchyma (Vectastain method; magni['. 35 x).

DISCUSSION

During the past decade, major advances have been made in the field of hepatocyte transplantation. How- ever, several issues need to be resolved before clinical trials of hepatocyte transplantation in humans can be carried out with a realistic chance o!-" success. One of these is determination of the optimal site for hepato- cyte implantation. Intrasplenic and other sites (kidney subcapsular, pancreas, thymus) are limited by the rel- atively small number of cells which can be transplanted and because heterotopic liver (auto)grafts undergo at- rophy (11,12). Intraperitoneal transplantation is also limited because of the necessity for introduction of var- ious types of matrix to provide a substratum for cell attachment and differentiation (2,3).

Intraportal transplantation seems to have many po- tential advantages including (i) transplanting hepato- cytes into the unique hepatic architecture, allowing for interaction with other hepatocytes and nonparenchy- mal liver cells; (ii) proximity to locally released growth factors; and (iii) the potential beneficial effects of portal-born hepatotrophic factors. In the past, direct portal infusion of isolated hepatocytes was associated with severe liver damage (5,10). In addition, early mor- tality resulted from aggregated material passing to the cardiopulmonary circulation, portal vein thrombosis,

and portal hypertension (5). All these together with in- ability to identify transplanted hepatocytes in the liver tissue, explains why the intraportal route has not been thoroughly tested.

Recently, we have demonstrated that after selective intraportal transplantation of a single-hepatocyte sus- pension, survival of rats was excellent and minimal his- tological injury was seen in the transplanted liver lobes (6). We postulated that early portal decompression through the nontransplanted liver lobes and minimal portal occlusion played a critical role. In this study, the results of selective intraportal hepatocyte transplanta- tion resembled those originally reported (6). Only some of the liver sections showed fibrotic lesions, which most likely followed resolution of microfocal necrosis caused by transplanted cells aggregates (13). The ELI SA findings, which have been confirmed by 10o70 SDS- PAGE and protein immunoblot data, revealed grad- ual and sustained increase in plasma albumin in Nagase analbuminemic rats transplanted with normal albumin producing rat hepatocytes (Fig. 2). Even more striking functional effect was seen in NAR rats transplanted re- peatedly through a subcutaneously placed indwelling vascular access device system connected to the portal vein tributary. Significantly, no liver tissue damage was seen on light microscopy, even though the total num- ber of hepatocytes transplanted was much larger than

242 Cell Transplantation • Volume 4, Number 2, 1995

(a)

(b)

Fig. 6. Analbuminemic rats subjected to repeated (6x) intraportal infusion of 5 x 10 6 normal rat hepatocytes showed a nor- mal liver architecture. (a) A large number of albumin-positive cells were forming (b) clusters containing up to 50 cells (Vec- tastain ABC; magnif. 30x and 60x, respectively).

that used in Group I rats. Histologically, both Group I and Group II rats had perfectly engrafted albumin- positive hepatocytes.

The results of our work follow the first report of hu- man gene therapy in which stable (18 too; duration of the treatment) partial correction of dyslipidemia has been achieved after intraportal infusion of autologous hepatocytes that were genetically corrected with recom- binant retroviruses carrying the LDL receptor (4). The

cells were infused through the catheter that was in- stalled at the time of partial hepatectomy. The post- transplant period was complication free. In particular, no evidence of portal vein thrombosis, portal hyper- tension and/or pulmonary embolism were found.

In conclusion, repeated intraportal infusions of iso- lated rat hepatocytes resulted in an excellent recipient survival, good cell engraftment, and remarkable func- tional effect with no apparent damage to the liver pa-

Hepatocyte transplantation • J.R. Rozoa ET AL. 243

renchyma. This method offers the option of repeating infusions if the number of transplanted hepatocytes was insufficient to obtain a desired functional effect, or if the transplanted ceils failed to survive or became rejected. It seems, that repeated administration of ceils via an implanted device might have a place in both ex vivo liver-directed gene therapy and in transplantation of allogenic isolated hepatocytes.

Acknowledgments-Supported by NIH grant #DK38763-06.

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