인공간에서 해독작용에 대한 관류속도의 영향

Theories and Applications of Chem. Eng., 2002, Vol. 8, No. 1

Theories and Applications of Chem. Eng., 2002, Vol. 8, No. 1

박영건1,2, 이와다 히로우1, 사이또우세이지3, 우에스기다케히코3, 이카이이와오3, 야마오카요시오3, 이카다요시히또1

1. 쿄토대학교 재생의공학연구소, 2. 전남대학교 의학연구소, 3. 쿄토대학 소화기내과

Effect of perfusate flow rate on the detoxification function of porcine hepatocytes in a bioartificial liver

Youeng Guen Park1,2, Hiro Iwata1, Seiji Satoh3, Takehiko Uesugi3, Iwao Ikai3, Yoshio Yamaoka3, Yoshito Ikada1

화학공학의 이론과 응용 제8권 제1호 2002년

1. Institute for Frontier Medical Science, Kyoto University, Japan 2. Research Institute of Medical Sciences, Chonnam National University, Korea 3. Department of Gastroenterological Surgery, Kyoto University, Japan

Theories and Applications of Chem. Eng., 2002, Vol. 8, No. 1

INTRODUCTION

The treatment of fulminant hepatic failure (FHF) promotes interest in the concept of temporary liver support system which increases the chance of survival by sustaining patients until donor liver is available. Several experimental devices have been obtained to prolong the viability and functions of cultured hepatocytes for higher metabolism [1]. Furthermore, the innovative membrane-based bioreactor design by Gerlach et al. which was composed of sinusoidal cell co-culture, and independent supply of oxygen and nutrients, and metabolite exchange has recently been proposed to maintain the stable metabolism over a period of 5 weeks [2]. However, so far, a few membrane designs have been studied in the clinical setting, and with little success. A laboratory scale device of good performance, with optimal operating condition is required. The mass transfer of oxygen, nutrients, toxins within the BAL that depends on the fluid dynamics and the transport properties of the membrane is an important factor to improve the functions of cultured hepatocytes. Oxygen depletion has been identified as the scale-limiting factor for animal cell cultures in hollow fiver bioreactors [3]. The maximum cell density from the analysis of oxygen concentration distribution depends on the fluid dynamics in the BAL system.

The assessment of efficiency of the in vitro BAL support device to apply the BAL treatment in FHF has been investigated theoretically and experimentally based on pharmacokinetics [4,5]. It can give a suitable basis for the optimal condition of the BAL and quantitative assessment of its metabolic functions. In this study, after an initial bolus loading, the effect of the perfusate flow rate on the function of the BAL was investigated using lidocaine. In addition, with a constant-rate infusion plus initial bolus loading test, the plateau level of lidocaine in the BAL was studied with increasing the perfusate flow rate.

METHODS AND MATERIALS

Bioreactor for a bioartificial liver: The methods of enzymatic liver digestion for the isolation of primary liver cells, modified method reported by Sielaff et al. [6], was established for pigs with about 12kg of body weight. Heparin sodium (Nippon Hoechst Marion Ltd., Tokyo) of 83 U/kg (body weight) was bolusly administered through the vein. A collagenase solution at 37℃ oxygenated by MENOX AL-2000, was circulated from the portal vein at 800 ml/min for approximately 20 min until the liver inspected by palpation was soft and well digested. After digestion, the liver capsule was incised on all lobes and hepatocytes were released into calcium and magnesium free Hanks balanced salt solution (HBSS) at 4℃. A hollow fiver cartridge (Duo-Flux M170D, JMS, Hiroshima, Japan) which is clinically used as a hemodialyzer was employed to prepare a bioreactor containing hepatocytes. The hollow fivers are made of cellulose diacetate with a nominal cut-off molecular weight of 32 kDa and an internal diameter of 195 □m. The volumes of the inner space and the shell space of the hollow fivers is 99 ml and 227 ml, respectively. The total volume of the circuit was 200 ml, except for the reservoir. The culture medium, consisted of 200 ml of the human whole blood diluted with 800 ml of Dilbeccos modified Eagles medium (DMEM) supplemented with 3.7g biocarbonate, 2,500 unit heparin, 40 unit insulin (Humulin R, lilly Japan, Kobe), 105 unit penicillin, and 0.1 g streptomycin, was perfused in a closed circuit through the luman of the BAL cartridge. The closed circuit was incubated in a flow culturing system (Hollow fiber reactor HFR-1000, EYELA, Tokyo, Japan) at 37℃. The shell portion of the cartridge was perfused at 200 ml/min and oxygenated with 95% air and 5% carbon dioxide gas mixture. 1000 unit of heparin was administered by bolus at every 24 hrs.

Lidocaine clearance test: For an initial bolus loading test, the lidocaine solution was injected by a bolus to the perfusate at a concentration of 10□g/ml from the port of the reservoir.      For a constant-rate infusion test, lidocaine was infused to the perfusate at constant-rate (R) with initial bolus loading (D). The drug infusion was continued until a steady state was reached. The lidocaine concentration was determined using TDX analyzer (Dainabot, Tokyo, Japan).

RESULTS AND DISCUSSION

Initial bolus loading of lidocaine (I-B): When a drug is administrated into the BAL to evaluate the function of the BAL, the lidocaine is removed by the process which includes adsorption and elimination. We can derive following equations under a well-stirred condition:

(1)

(2)

where V1 and V2 are the volumes of the reservoir and the BAL cartridge, respectively, and C1 and C2 are the concentrations of lidocaine in the reservoir and the BAL cartridge, respectively. CL, Q, and R are the clearance and the perfusate flow rate, and the constant rate of drug, respectively. In case R is equal to zero, the change of lidocaine concentration in the reservoir with initial conditions is solved as follows

(3)

Initial conditions:

Eq. (3) represents the concentration change of the drug with time which has two mechanisms such as the adsorption (□) and the elimination (□) phases. The clearance can be expressed under the well-stirred condition by

(4)

where CLint relates the intrinsic rate of metabolism to the unbound toxin concentration at enzyme sites. Eq. (4) predicts that CL increases with the increasing of Q and approaches to a maximum value, that is, CLint. Under the same CLint, Fig. 1 shows the effect of Q on the concentration-time profile of lidocaine concentration for I-B test. As were expected, with increasing Q, the concentration-time curve of lidocaine drops rapidly due to the increase of □. The figure shows the increase of Q enhanced the function of the BAL.

Eq. (4) is rewritten using the extraction ratio (E).

(5)

The value of E from Eq. (5) represents the efficiency of the function of the BAL for perfusion, which is the extraction ratio. In case of human liver, the values of E and Q are 0.64 and 1,500 ml/min, respectively. The values of E for human liver at different perfusate flow rates were obtained from Eq. (5) under the same CLint value. The efficiency of the BAL increased with increasing CLint, but decreased with increasing Q. Under Q of 200ml/min, the extraction ratios of our BAL were 0.06~0.19 which are very small in comparison with human liver (0.93). CLint corresponding to perfusate flow rate of 200ml/min for our BAL was 30 ml/min on average, which is 15% of perfusate flow rate. It means that the efficiency of the BAL is 20% of that of human liver. If the BAL system has the lower metabolic function, the device works with limited mass transport due to lower efficiency even though the perfusate flow rate was infinite in the BAL cartridge.

Constant-rate infusion with initial bolus loading of lidocaine (R□0, C-I-B): When a lidocaine solution is infused at a constant-rate with an initial bolus administration, the lidocaine concentration-time behaves as the sum of both a constant-rate infusion and an initial bolus loading. When the lidocaine is administrated with R/□ as an initial bolus loading, the the time to approach the plateau value (t97%) was about 7 hrs. D under R =40□g/min was 0.11mg calculated from □ of 0.3633 min-1 determined from previous experiment, I-B test. The concentration of lidocaine decreases with time, reaches a minimum, increases slowly and approaches the plateau value. On the other hand, When the lidocaine is administrated with R/□ as an initial bolus loading, t97% was 8.6min. The initial bolus amount, 4.35mg under R = 40□g/min, was determined from previous experiment of initial bolus loading. The concentration of lidocaine drops and then reaches the plateau level rapidly. The initial bolus drug amount determined from previous experiment is important to achieve the plateau level rapidly with C-I-B, and must be administrated at the level of R/□.

The plateau level of lidocaine obeying the first-order reaction depends on the perfusate flow rate. Under the same CLint of the BAL, Fig. 2 shows the effects of Q on the plateau level of lidocaine for C-I-B and D=R/□. Initial bolus loading experiment for Q =200 ml/min was carried out to obtain  before C-I-B test. For Q of 25 and 100 ml/min, the values of D were obtained from □ determined using Eq. (4). Smaller amount of drugs were injected with the greater blood flow rate due to the increase of  with increasing Q. The lidocaine concentration varies with combinations of parameters such as the perfusate flow rate and the initial bolus loading. With increasing the perfusate flow rate, the plateau level of lidocaine decreased as seen in Fig. 2. The time to reach 95% percent of the asymptotic lidocaine concentration was almost the same regardless of Q. The time to reach the plateau level is occurred due to the difference of the distribution phase between the initial bolus loading (□ and □ phases) and the constant-rate infusion (□ phase). Without the distribution phase, the plateau level will be instantaneously reached.

CONCLUSION

As for the mass transport of chemicals, the design of the optimal BAL device is to ensure the successful detoxification of toxins produced endogenously in the body and the long term maintenance of cell function in the BAL cartridge. Oxygen supply is one of the limiting factors that determine liver survival and functions. The function of the BAL can be maintained for a long term without limitation in the mass transfer of the required oxygen from the fluid to the cell. The maximum mass transport of chemical in the BAL is important factor for detoxification of toxin as well as oxygen supply. In this study, the effect of perfusate flow rate on the function of the BAL was verified by pharmacokinetics analysis with I-B and C-I-B. In conclusion, the increase of the perfusate flow rate enhanced the function of the BAL and decreased the plateau level of toxin. This work is useful to study the optimal detoxification condition of drug within the BAL.

REFERENCES

1. S.L. Nyberg et al., Biotechnol Bioeng. 41 (1993) 194

2. J. Gerlach et al., Int J Artif Organs. 17 (1994) 301

3. A. Bader et al., Artif Organs. 19 (1995) 368

4. Y. G. Park, H. Iwata, and Y. Ikada, Annals of the New York Academy of Sciences 944 (2001) 296

화학공학의 이론과 응용 제8권 제1호 2002년

5. Y. G. Park et al., Artificial Organs, in press (2002)