Comparative analysis of preparation of human hepatocytes by the ethylenediamine tetraacetic acid and collagenase technique

Purpose: To compare the viability of human hepatocytes dissociated by the ethylenediaminetetraacetic acid and collagenase techniques. Methods: Hepatocytes were prepared by dissociation of liver fragments obtained from hepatectomies performed for therapeutic purposes at the Service of Digestive Tract Surgery, Federal University of Triângulo Mineiro. Results: During the first 4 days of the experiment, 70% of the cells presented birefringent membranes and were not stained with 2% erythrosine, and were therefore considered to be viable. During the first 3 days, hepatocyte viability was on average 71% in the EDTA group and 76% in the collagenase group, with no significant difference between groups. No significant difference was observed between groups at any time. The secretion of albumin by the cultured hepatocytes was preserved up to the seventh day. Mean albumin secretion during the first 3 days was 50 μg/ml in the two groups and a reduction of albumin production was observed from the fourth to the seventh day. Again, no significant difference was observed between groups at any time. Conclusion: Cell viability and preservation of albumin secretion by hepatocytes are similar for the EDTA and collagenase techniques.


Introduction
Several research centers have been searching for auxiliary liver transplantation techniques either for definitive use in chronic or temporary situations or for acute situations, thus prolonging the waiting time for functional improvement of the regenerated liver or for appearance of a compatible liver transplant donor, with the latter certainly continuing to be the only therapy for end-stage hepatic insufficiency and metabolic disorders.The great expectation of finding a bioartificial liver has directed research at the utilization of isolated viable hepatic cells with preserved functions 1,2,3 .Sun 4 , using a semipermeable alginate-polylysine membrane for the microencapsulation of hepatocytes, reported satisfactory viability of the cells in vitro and in vivo 5 .Several investigators studying the preservation of hepatocyte function in homozygous Gunn rats noted a significant reduction of bilirubin levels over a period of 30 days when intraperitoneally transplanted microencapsulated hepatocytes were used 6 .Immunoprotection offered by the microcapsule membrane, as well as a significant reduction of bilirubin levels, has been demonstrated in Gunn rats receiving alginate-polylysinemicroencapsulated hepatocytes 6 .The most commonly used methods for the dissociation of hepatocytes from other types of cells (biliary cells, supporting cells, vascular cells, etc.) without compromising their metabolic functions are EDTA (ethylenediamine tetraacetic acid) or collagenase perfusion, or a combination of both.Therefore, the objective of the present study was to compare the viability of human hepatocytes dissociated by the EDTA and collagenase techniques.

Methods
To compare the dissociation of hepatocytes by the EDTA and collagenase techniques, hepatocytes were prepared by dissociation of liver fragments obtained from hepatectomies performed for therapeutic purposes at the Service of Digestive Tract Surgery, Federal University of Triangulo Mineiro (UFTM).All patients gave free informed consent to participate and the study was approved by the Ethics Committee of UFTM.

Dissociation of human liver fragments by the EDTA method
Hepatocytes were prepared by dissociation of a liver fragment with EDTA.The perfusion solution had the following composition 7 : 140 mM NaCl, 5 mM KCl, 0.8 mM MgSO 4, 1.6 mM Na 2 HPO 4, 0.4 mM KH 2 PO 4, 2 mM EDTA, and 25 mM NaHCO 3 .The EDTA solution was perfused through small catheters placed in the venous segments and fixed with 4.0 Prolene sutures at a constant temperature of 37ºC.A perfusion pump was used to guarantee continuous perfusion for 30 to 40 min at a constant flow rate of 3 to 4 ml/kg liver fragment/min.After perfusion, the liver fragment was placed in a 50-ml sterile glass container (Becker) filled with 20 ml HAM"S 12 culture medium at 4ºC and carefully cut until only small fragments were left.This suspension was then filtered through gauze bandage.The suspension obtained was ultracentrifuged twice at 5000 rpm for 2 min.The supernatant was aspirated and discarded and the cell mass obtained was weighed.Hepatocytes were separated on a pressure gradient by adding one volume of Percoll solution at a concentration of 1/10, using a proportion of 1 g cell mass for each 4 ml of Percoll solution.This solution was centrifuged at 6000 rpm for 5 min.Hepatocytes were counted in a Nageotte chamber.Cell viability was evaluated by determining membrane birefringence and by the 2% erythrosine exclusion test, and the results are expressed as percentage of viable cells.

Dissociation of human liver fragments by the collagenase technique
The method of in situ perfusion with collagenase described by Berry and Friend 8 and modified by Guilouzo 9 was used, which differs from the EDTA method in terms of the solutions employed and the time of perfusion.Perfusion solution -washing at a flow rate of 30 ml/min for 15 min: 160.8 mM NaCl, 3.15 mM KCl, 0.

Hepatocyte culture
The cells obtained with the two dissociation techniques were cultured in medium consisting of mixture of 75% Williams E (Gibco) and 25% Leiba 15 medium enriched with 100 mg/ml bovine serum albumin, 0.1 U/ml insulin, 30 IU/ml crystalline penicillin G, 100 mg/ml streptomycin, and 10% fetal bovine serum.The pH was adjusted to 7.4 with 2.2 mg/ml sodium carbonate.After 4 to 6 h of incubation at 37ºC in a humid atmosphere (95% air and 5% CO 2 ), the hepatocytes were found to be adhered to the bottom of the plate.The culture medium was then aspirated and replaced with the same fresh medium supplemented with hydrocortisone semisuccinate at a proportion of 4.5 x 10 5 mg/ml.This medium was changed daily until the end of the experiment.The hepatocytes obtained were cultured on 5-cm plastic Petri dishes in medium at a proportion of 1.5 x 10 5 cells/ cm 2 .Twenty-eight Petri dishes, including 14 for hepatocytes obtained by EDTA dissociation and 14 for hepatocytes obtained by the collagenase technique, were used.

Analysis of the preservation of albumin secretion by hepatocytes obtained with the EDTA and collagenase methods
The presence of human albumin was determined in the culture supernatant obtained daily from the 28 Petri dishes.Albumin was assayed in the aspirated culture medium using an antigen specific for the detection of human albumin, thus preventing contamination with the bovine albumin used in the preparation of the culture medium.

Results
Hepatocyte viability was compared daily in two plates from each group for a period of 7 days.The results showed that, during the first 4 days of the experiment, 70% of the cells presented birefringent membranes and were not stained with 2% erythrosine, and were therefore considered to be viable.During the first 3 days, hepatocyte viability was on average 71% in the EDTA group and 76% in the collagenase group, with no significant difference between groups.An expressive reduction of cell viability was observed after the fifth day for both techniques, affecting the rates of viable cells.No significant difference was observed between groups at any time (Figure 1).The secretion of albumin by the cultured hepatocytes was preserved up to the seventh day.Mean albumin secretion during the first 3 days was 50 µg/ml in the two groups and a reduction of albumin production was observed from the fourth to the seventh day.Again, no significant difference was observed between groups at any time (Figure 2).organ.There are still no standardized liver dissociation techniques; however, all techniques propose the perfusion of solutions through the portal vein 5 in intact livers or small liver fragments 11,12 , with the methods differing in terms of the solution used for perfusion witch EDTA 7 , hyaluronidase, collagenase or a combination of these solutions 13 .In the present study, cell viability was 85% and 83% after dissociation with EDTA and collagenase, respectively, rates higher than those reported by other investigators 14 .Up to the third day, mean viability was found to be similar for the two techniques and a marked quantity of viable hepatocytes was observed, which might be used in hemodialyzers or in studies on new drugs or cellular genetic modifications 2,9,15 .The present results were similar to those reported in previous studies employing in situ dissociation of rat livers.Cell viability was excellent and no difference was observed between dissociation with EDTA and collagenase.We therefore recommend the use of EDTA as a dissociation solution because of its much lower cost compared to collagenase.Another advantage of EDTA is that its action is interrupted at the end of dissociation, in contrast to collagenase, thus preserving cellular functions.

Conclusion
Based on the present experiment we conclude that cell viability and preservation of albumin secretion by hepatocytes are similar for the EDTA and collagenase techniques.

Discussion
Liver transplantation has undergone great advances since 1963 when Starzl in the United States proposed homotransplantation of the liver in humans, a procedure which continues to be the only effective technique for functional replacement of the liver until today.Despite the advances in transplantation techniques, better immunosuppressive agents and improved care during postoperative recovery, 20 to 30% of patients with hepatic insufficiency continue to die while on the waiting list, mainly because of the small number of donors.In this respect, over the last few years, the development of a bioartificial liver or of viable hepatic cell transplants has raised the interest of several research centers in an attempt to temporarily replace the failing liver, maintaining the patient alive until a compatible donor is found, or even to replace treat isolated metabolic disorders 10,11 .To obtain isolated cells with preserved functions, it is necessary to dissociate the hepatocytes from the liver to separate them from biliary, vascular and connective tissues which are often responsible for rejection of the 7 mM Na 2 HPO 4 .12H 2 O, 33 nM Hepes (N-[2-hydroxyethyl] piperazine-N-[2-ethanesulfonic acid]).Perfusion solution -dissociation at a flow rate of 15 ml/min for 20 min: 160.8 mM NaCl, 3.15 mM KCl, 0.7 mM Na 2 HPO 4 .12H 2 O, 33 mM Hepes, 0.075 mM CaCl 2 , and 0.05% collagenase D.

FIGURE 1 -
FIGURE 1 -Viability of isolated cultured hepatocytes obtained by EDTA and collagenase dissociation.