Fructose protects rat hepatocytes against hypoxic injury during the process of isolation and microencapsulation

Fructose Protects Rat Hepatocytes Against Hypoxic Injury During the Process of Isolation and Microencapsulation C.H. Yu, X.S. Leng, J.R. Peng, Y.H. Wei, J.C. Liu, and R.Y. Du

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ICROENCAPSULATION of cells within synthetic semipermeable membranes is a novel technique that enables the transplantation of cells without the need for the use of immunosuppressants. Experimently microencapsulated hepatocytes have been successfully used in the treatment of acute liver failure1–3 and congenital liver enzyme deficiences such as bilirubin-uridine diphosphate deficience of the liver (Gunn rat).4 – 6 This treatment demands a large quantity of healthy nude hepatocytes or microencapsulated hepatocytes at one time. To date, however, apparatus for microencapsulation are of low efficiency, the isolation and microencapsulation usually is a time-consuming process. During this time, hapatocytes are under the anoxic or hypoxic situation while being embedded in the highly viscous mixture of alginate. Hepatocytes are far more sensitive to hypoxia than other cells and easily injured. Therefore, measures protecting liver cells against damage during isolation and microencapsulation are important. Hypoxia is mainly characterized by a loss of the oxidative phosphorylation capacity of the mitochondria and decreased ATP levels.7–11 The fermentation pathways that operate in facultative anaerobes during oxygen deprivation12 are restricted in mammalian cells. Thus at low PO2, ATP is only supplied by way of anaerobic glycolysis. Cellular integrity may depend on the availability of glycolytic substrates8,13 and their intracellular reserves. Fructose is a well-known glycolytic substrate that protects the cells when synthesis of ATP is compromised.14,15 Cells could produce F-1-P and loaded with it when enough fructose was supplied before hypoxia. It has been reported that fructose can protect hepatocytes against the damage of hypoxia or anoxia.16 To the best of our knowledge, there is no report about the protective effect of fructose in the microencapsulation process. We investigated the protective effect of fructose in the process of isolation and microencapsulation of rat liver cells.

MATERIALS AND METHODS Animals Male Sprague-Dawley rats weighing 250 to 300 g were supplied by the animal center of Beijing Medical University (Beijing, China) and housed in individual cages under room temperature of between 20 to 28°C. Rats were allowed free access to chow and water. 0041-1345/99/$–see front matter PII S0041-1345(98)02101-0

Isolation of Hepatocytes Hepatocytes from Sprague-Dawley rats were isolated by a modified in situ portal vein perfusion (two steps) technique described by Seglen.17 The system of perfusion consists of a thermostatic bath (Cole, Parmer, Ill) in which the perfusion solution is bathed and a perfusion pump impels the solution by a catheter cannulated in the portal vein of the rats. Solutions used for washing and perfusion are solution I, composed of 300 mL of Ca21 and Mg21-free Hank’s with 20 mM fructose (Merck Co.) and 50 mM EGTA (Sigma Chemical Co, St. Louis, Mo) pH 7.4; and solution II, composed of 100 mL of Hank’s with 0.05 g type IV collagenase (Sigma), 18 mM HEPES (Sigma), and 20 mM fructose, pH 7.4. Animals were intraperitoneally anesthetized with sodium pentobarbital (60 mg/kg body weight) and given 2000 IU of sodium heparin to prevent coagulation of blood during cannulation. After the abdomen was opened, the inferior vena cava was tied above the renal veins. The portal vein was cannulated with a 16-gauge intravenous plastic cannula (Becton Dickinson Vascular Access, Sandy, Utah). The liver was irrigated with solution I to expel the blood. The solution was kept at 37°C and oxygenated by bubbling through a mixture of O2 1 CO2 (95%:5%). The solution was pumped through the liver at the rate of 15 mL/min for 20 minutes, and the perfusate was drained away through an incision made at the right atrium. After the liver was washed free of blood, another 18-gauge cannula was inserted into the inferior vena cava through the right atrium to the level of the hepatic vein and tied in place. Then the liver was perfused with solution II for 7 to 8 min. These enzymes were conserved by recycling the effluent to a reservoir that was immersed in a water bath that kept the solution at 37°C, and solutions were oxygenated by bubbling through a mixture of O2 1 CO2 (95%:5%). After perfusion, the liver was swollen with blebs and oozing of solutions on the surface. At this point, the liver was cut away from the abdomen with scissors and forceps. The liver was placed on a Petri dish with 20 mL solution II. The Glisson capsule was stripped off, and the hepatic parenchyma combed down. The resulted suspension was filtered through a 95-m m sieve to remove cell clumps and connective tissue debris. The partially purified hepatocytes suspension was then differentially centrifuged (centrif-

From the Department of Hepatobiliary Surgery, People’s Hospital, Beijing Medical University, Beijing, China. Supported by National Natural Science Fundation of China (Special allocate fund, 1997). Address reprint requests to Dr Leng Xisheng MD, Department of Hepatobiliary Surgery, People’s Hospital, Beijing Medical University, Beijing 100044, China. © 1999 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010

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FRUCTOSE PROTECTS RAT HEPATOCYTES AGAINST INJURY ugal force-50g) at 4°C to wash the hepatocytes with Hank’s three times. Hepatocytes yield routinely varied between 1.5 and 2.5 3 108 cells per liver with a viability rate varying from 85% to 92.5% as determined by means of trypin blue exclusion.

Preperation of Microsphere of Hepatocyte-Sodium Alginate and Microencapsulated Hepatocytes Isolated hepatocytes were microencapsulated by an ultra-thin sodium alginate-poly-L-lysine-sodium alginate copolymer membrane. The technique is a modification of the procedure initially described by Lim18 and subsequently by O’Shea, Goosen, and Sun.19 Briefly, isolated hepatocytes were suspended in a mixture of 2% (viscosity 5 266 cps) sodium alginate (Sigma). The mixture was then extruded through a droplet-generating apparatus to form microdroplets of approximately 300 to 700 m m in diameter. The alginate microdroplets were reacted with 0.1% poly L-lysine (Sigma) for exactly 15 minutes to facilitate the formation of an outer skin of polylysine of the surface of the alginate microdroplet. The polylysine is covalently bonded to the alginate exposed on the surface of the microdroplet. The polylysine-coated alginate microdroplets were further reacted with a 0.2% solution of sodium alginate to form covalent linkages between the polylysine and the alginate. (In the microsphere of hepatocyte-sodium alginate used for viability test, poly L-lysine was replaced by 0.9% salt solution.) Finally, the capsules were liquified by 0.05 M sodium citrate, pH 7.4, for 6 minutes, and alginate was partially removed by 0.9% sodium chloride washing. Each milliliter of microencapsulated hepatocytes contained approxmately 1 3 107 hepatocytes.

1081 Table 1. Viabilities of Hepatocytes in Three Groups of Rats Group

N

Before Microencapsulation

After Microencapsulation

A B C

6 6 6

91.25% 6 16.15% 90.2% 6 11.65% 89.2% 6 16.4%

87% 6 10.5% 78.2% 6 12.5% 76.9% 6 15%

A vs B and C, P , .001 (t test).

in culture medium (IU/L)/LDH in culture medium (IU/L) 1 LDH in cell pellets (IU/L).

Function of Protein Synthesis Total protein of microencapsulated heptocytes in culture medium was examined for 3 consecutive days by autochemical analyser.

Statistics The results were expressed as mean 6 SEM in three groups. Data were analyzed with t test or Q test.

RESULTS Viability of Hepatocytes in Different Groups

Although the viability was generally decreased to some extent, group A (added with fructose of 20 mmol/L in perfusions) had a higher rate of viability than other groups after a process of isolation and microencapsulation (Table 1); P , .001 (t test).

Study Design

The Time of Handling in the Process

Sprague-Dawley rats were divided into three groups of six rats each. They were fasted overnight before the experiment. Perfusion was carried out by using solution I and solution II in group A but perfusion solution contained no fructose in groups B and C. The time of the whole process in each rat was counted from the very beginning of the isolation to the end of microencapsulation.

Handling time was 5.1 6 0.57 hours in group A, 5.2 6 0.34 hours in group B, and 5.2 6 0.56 hours in group C. Q test shows that differences are not significant, P . .05.

In Vitro Culture Microencapsulated hepatocytes (1 3 107) were cultured with 10 mL culture medium in a humidified atmosphere composed of 5% CO2 in air at 37°C. The culture medium used in the three groups was RPMI 1640 supplemented with 18 mmol/L HEPES (Sigma), streptomycin 100 m g per mL, penicillin 100 IU per mL, gentamicin 100 m g per mL, insulin 0.5 unit per mL, and dexamthasone 1027 M. Fructose (10 mmol/L) was added only in group C. The medium was refreshed daily. The collected medium and microencapsulated hepatocytes were stored at a temperature of 220°C for biochemical assays.

Viability Test After resolving the alginate-hepatocytes microdroplets that were used for viability test by 0.05 mmol/L sodium citrate, we examined viability of hepatocyte by means of trypan blue exclusion (0.4% 1:1).

Lactate Dehydrogenase Leakage We measured lactate dehydrogenase (LDH) activity in both the culture medium and microencapsulated hepatocytes by autochemical analyser (Shimadzu CL-7000 Japan). LDH leakage (%) 5 LDH

LDH Leakage

Table 2 shows LDH leakage after 24 culture hours. Group A has far less leakage than groups B and C, P , .001 (t test). Protein Synthesis

Figure 1 compared the three groups in their total protein output daily in culture medium in the first 3 consecutive culture days after handling. The damage of protein synthesis was less serious in group A than in groups B and C. Q test shows the difference was significant, P , .01. DISCUSSION

Under the situation of hypoxia, cells lose their capacity of mitochondrial oxidative phosphorylation. ATP production Table 2. The LDH Leakage of Hepatocytes in Three Groups of Rats After 24 Hours Culture Group

N

LDH Leakage (%)

A B C

6 6 6

25.3% 6 5.43% 45.2% 6 16.17% 47.2% 6 13.3%

A vs B and C P , .001 (t test). LDH, lactate dehydrogenase.

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was better than in groups B and C (P 5 .001), an evidence of the protective effect of fructose in hypoxia. LDH leakage is an index of the integrity of a cell’s membrane. Enough F-1-P loading provided the cells with ATP during hypoxia and maintained stable condition of the cellular ionic gradients. Our results showed that cytoplastic enzyme release (LDH leakage) in group A was lower than the other two groups. The differences were significant. Protein synthesis, a major metabolic function of liver cells, was very sensitive to the changes in pO2 levels and was generally reduced during the process of a cell’s isolation and encapsulation. But our results showed that it was protected by the addition of fructose in perfusate. The reduction of protein synthesis in group A was less than groups B and C. It indicated that fructose could supply ATP to maintain the function of protein synthesis to some extent during hypoxia. Fructose added in medium after microencapsulation (group C) showed no protection. This result is similar to other’s16 because hepatocytes were not pretreated with fructose before hypoxia so there was not enough F-1-P in cells and not enough ATP supply in the stress of hypoxia. Taken together, the experimental data reported here indicate that perfusion with an oxygenated solution containing fructose improves the recovery of the hepatocytes functions during the process of isolation and microencapsulation. Fig 1. Effect of fructose on protein synthesis of microencapsulated hepatocyte after microencapsulation in three of groups of rats. Perfusion solution contained fructose (20 mmol/L) in group A but there was no fructose in groups B and C. Microencapsulated hepatocytes were cultured under the aerobic condition. Culture medium was added with fructose (10 mmol/L) only in group C. At the indicated times, aliquots of culture medium were taken and total protein measured as described in the text. Results were expressed as mean 6 SEM of six separate determinations. Q test showed a significant difference, P , .01 (Group A vs B and C).

is decreased, the cell membranes are damaged, and cells lose their function.20,21 At this condition, the metabolisms of cells are changed rapidly.8 –11,13,22,23 The ATP production mainly depends on glycolysis. So, the severity of the hypoxia-mediated damage has been correlated with glycolytic substrates in the cells before hypoxia.11,23–26 Preincubation of cells with fructose under the aerobic condition before the onset of hypoxia offers such a protection because of F-1-P loading. It has been reported that hepatocytes received fructose during 10 minutes of aerobic incubation and when further incubated under hypoxia, F-1-P production accumulated in amounts sufficient to compensate for the ATP requirement of hypoxic cells.16 From our results, the handling process was fairly long from isolation to microencapsulation, averaging 3 hours or more per rat. During this time, hepatocytes are under the anoxic or hypoxic situation (cells were embedded in viscous alginate mixture). Viability of cells was decreased significantly. But cell viability in group A (added with fructose 20 mmol/L)

ACKNOWLEDGMENTS The authors thank Dr A.M. Sun, Department of Physiology, Faculty of Medicine, University of Toronto, for introduction and help in the technique of microencapsulation and hepatocytes isolation.

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