An in situ perfusion protocol of rat epididymal adipose tissue useful in metabolic studies

Experimental approaches involving the perfusion of tissues and organs offer the advantage of improved physiological relevance over the use of isolated tissues or cells while at the same time being much more controlled and tissue-specific than studies in vivo. Nevertheless, there have been few metabolic studies performed in perfused white adipose tissue, largely because of the difficulty of the surgical technique involved. Although some methods have been described, they are difficult to use as perfusion protocols and their reproducibility is poor. We have modified a rat perfusion method, based on a modification of the Ho and Meng technique, for use with epididymal white adipose tissue (eWAT), and we present it here as a protocol to be reproduced. We also offer surgical solutions for the most common variants of vessel distributions in rats. Using the protocol described here, the perfused adipose tissue is viable and metabolically active, as indicated by the maintenance of tissue ATP levels and adiponectin secretion and by endogenous lipolysis regulation. Moreover, there is a high level of lipoprotein lipase activity in the endothelium of the tissue, which is heparin-releasable. Thus, this method is a useful and reproducible tool that allows the perfusion of eWAT for use in metabolic studies.     

Ex Situ Normothermic Machine Perfusion of Donor Livers

In contrast to conventional static cold preservation (0-4 °C), ex situ machine perfusion may provide better preservation of donor livers. Continuous perfusion of organs provides the opportunity to improve organ quality and allows ex situ viability assessment of donor livers prior to transplantation. This video article provides a step by step protocol for ex situ normothermic machine perfusion (37 °C) of human donor livers using a device that provides a pressure and temperature controlled pulsatile perfusion of the hepatic artery and continuous perfusion of the portal vein. The perfusion fluid is oxygenated by two hollow fiber membrane oxygenators and the temperature can be regulated between 10 °C and 37 °C. During perfusion, the metabolic activity of the liver as well as the degree of injury can be assessed by biochemical analysis of samples taken from the perfusion fluid. Machine perfusion is a very promising tool to increase the number of livers that are suitable for transplantation.     

Engineering organ perfusion protocols: NMR analysis of hepatocyte isolation from perfused rat liver

The development and use of an extracorporeal liver support device depends upon the isolation of a large number of viable, functioning hepatocytes from whole or partial livers. Current practice, however, produces nonoptimal yields, given that a large percentage of hepatocytes initially present are not successfully isolated. The normal hepatocyte isolation protocol consists of sequential perfusion with calcium chelating and collagenase buffers, and then separation of viable hepatocytes from non-viable and nonparenchymal cells, usually on the basis of cell density. In order to improve understanding regarding the metabolic and perfusion state of the liver during this perfusion protocol, ATP, pH, and tissue perfusion were evaluated using nuclear magnetic resonance (NMR). Perfusion with calcium chelating buffer was found to have minimal effect on the metabolic and perfusion parameters, whereas subsequent perfusion with collagenase buffer produced large declines in ATP, pH, and homogeneity of perfusion within 3 min. Perfusion with calcium-chelating buffer alone, or perfusion with calcium chelating buffer followed by a short period of ischemia to mimic the perfusion disruption of collagenase, did not produce the same decline in metabolic parameters. This NMR data suggested that enhancing the early perfusion and penetration of collagenase or prolonging the nontoxic calcium-chelation step may improve the yield and/or functionality of isolated cells. Therefore, several altered perfusion protocols were evaluated in terms of yield of viable parenchymal hepatocytes and hepatocyte albumin production. Although increasing the perfusion flow rate and initial perfusion with inactive (cold) collagenase did not produce significant improvements when compared with the control protocol (control cell yield 226 +/- 42 x 10(6) viable hepatocytes for 10- to 14-week-old female Lewis rat), prolonging and enhancing the calcium-chelating perfusion step or increasing the collagenase concentration did yield a significantly great number of viable parenchymal hepatocytes (393 +/- 44 and 328 +/- 39 x 10(6) viable hepatocytes, respectively) with no change in albumin production per seeded viable cell. (c) 1994 John Wiley & Sons, Inc.     

分离小鼠肝细胞的一种简易灌流法

我们设计了一种分离小鼠肝细胞的简易灌流法。它具有操作简便,不需特殊装置,胶原酶耗费少等优点。在总活细胞产量及每100g体重活细胞产量等方面显著高于振荡法(P<0.001),而接近Seglen灌流法。此外,对经本法分离的肝细胞及肝非实质细胞进行培养,获得了良好效果。     

Impaired therapeutic vasculogenesis by transplantation of OxLDL-treated endothelial progenitor cells

Previous in vitro studies have revealed that oxidized low density lipoprotein (OxLDL) has negative effects on the proliferation and activity of endothelial progenitor cells (EPCs). Here, we evaluated the effect of OxLDL on the therapeutic potential of EPCs in ischemia-induced neovascularization. EPCs derived from mobilized human peripheral blood mononuclear cells were cultured without or with OxLDL before transplantation. Hindlimb ischemia models were surgically induced in athymic nude mice, which then received an intracardiac injection of 3 x 10(5) EPCs. By laser Doppler perfusion image and ischemia damage score, we found that blood perfusion and ischemia damage were less well recovered in the OxLDL-treated EPC transplantation group than in controls. Histological examination showed fewer transplanted EPCs and lower capillary density in ischemic tissue. Local delivery of Stromal cell-derived factor (SDF-1) restored this defect and improved blood perfusion by recruiting OxLDL-treated EPCs to the ischemic area and increasing host capillary density. These results provide for the first time direct evidence that OxLDL impaired the therapeutic potential of EPCs in ischemia-induced neovascularization through an inhibitory effect on the migration, adhesion, and incorporation of EPCs into vasculature and/or entrapment in the perivascular region in vivo. A therapeutic strategy based on SDF-1 administration ameliorated such defects and improved postischemic neovascularization.     

CT对于肝脏良性占位性病变及肝癌的鉴别诊断价值分析

目的:探讨CT对于肝脏良性占位性病变及肝癌的鉴别诊断价值。方法:收取2013年3月至2016年3月我院收治的肝脏良性占位性病变及肝癌患者101例作为研究对象,按照病变类型将其分为A、B、C三组。其中A组包含原发性肝癌患者32例,B组包含肝转移癌患者28例,C组包含肝血管瘤患者41例。采用CT全肝灌注扫描模式对三组患者占位病灶组织、病灶周围组织及正常肝脏组织灌注参数进行比较。结果:三组占位病灶组织,B组患者肝动脉灌注量(HAP)最低,C组患者HAP最高;A组患者门静脉灌注量(PVP)最低,C组患者PVP最高,三组两两比较均有显著差异(P0.05)。C组总肝灌注量(TLP)明显高于A组和B组(P0.05),A、B组间无统计学差异(P0.05)。三组肝动脉灌注指数(HPI)无明显差异(P0.05);B组病灶周围组织HAP及HPI明显高于A、C组(P0.05),A、C组间无统计学差异(P0.05);三组PVP及TLP差异不显著(P0.05);三组正常肝脏组织CT灌注参数均无显著差异(P0.05)。结论:CT灌注成像对于原发性肝癌、肝转移癌及肝血管瘤具有一定的鉴别诊断价值,但明确诊断仍需结合其他检测方法进行。     

Distribution of chylomicron degradation products in the isolated,perfused rat heart

Chylomicron degradation by hearts from fed and fasted rats was studied using a perfusion technique, which allows the separate collection of coronary (Q_rv) and interstitial effluent (Q_i). Upon perfusion with [³H]-cholesterol-containing chylomicrons the tissue recovery of label was highest in the fasted state, while label recovered in Q_i was highest in the fed state. Density gradient centrifugation of Q_i indicated that the label was recovered in lipoproteins with higher densities: low density lipoproteins (1.019<d<1.050), high density lipoproteins (1.050<d<1.21) and a fraction of d>1.21. These particles probably represent chylomicron degradation products (remnants and “surface fragments”). Our results indicate that tissue cholesterol uptake during chylomicron degradation may be inhibited in the fed state. Furthermore, the role of the myocyte (or interstitial) lipoprotein lipase in chylomicron degradation is discussed.     

Selective evaluation of high density lipoprotein from mouse small intestine by an in situ perfusion technique

The small intestine (SI) is the second-greatest source of HDL in mice. However, the selective evaluation of SI-derived HDL (SI-HDL) has been difficult because even the origin of HDL obtained in vivo from the intestinal lymph duct of anesthetized rodents is doubtful. To shed light on this question, we have developed a novel in situ perfusion technique using surgically isolated mouse SI, with which the possible filtration of plasma HDL into the SI lymph duct can be prevented. With the developed method, we studied the characteristics of and mechanism for the production and regulation of SI-HDL. Nascent HDL particles were detected in SI lymph perfusates in WT mice, but not in ABCA1 KO mice. SI-HDL had a high protein content and was smaller than plasma HDL. SI-HDL was rich in TG and apo AIV compared with HDL in liver perfusates. SI-HDL was increased by high-fat diets and reduced in apo E KO mice. In conclusion, with our in situ perfusion model that enables the selective evaluation of SI-HDL, we demonstrated that ABCA1 plays an important role in intestinal HDL production, and SI-HDL is small, dense, rich in apo AIV, and regulated by nutritional and genetic factors.     

电针对小鼠肝脏血流灌注量影响的激光散斑成像显示

目的：应用激光散斑成像技术连续监测电针过程小鼠肝脏表面血流灌注图像,研究电针不同穴位对肝脏血流灌注量的影响,探讨激光散斑技术在针灸效应研究中的应用价值。方法：采用Moor．FLPI激光散斑血流成像系统分别对足三里组、曲泉组、非经非穴组正常小鼠电针30min以及不电针对照组连续观察30min过程中肝脏表面血流灌注量变化进行观察,分析电针不同穴位、各个时点肝脏血流变化的规律。结果：（1）肝脏激光散斑图显示电针后各电针组肝脏表面整体血流灌注均增加,肝门附近区域灌注量增加幅度大于肝脏边缘区域;（2）电针各时点各电针组肝脏血流灌注量均出现增加,电针0～20min灌注量增加率为足三里组〉曲泉组〉非经非穴组;电针25～30min为足三里组〉非经非穴组〉曲泉组。结论：激光散斑血流成像技术能够精确记录显示电针过程肝脏表面的微循环变化情况,电针可以增强正常小鼠肝脏血流灌注量,电针增加肝脏血流灌注的效应存在穴位特异性。     

Hepatic denervation impairs the assembly and secretion of VLDL-TAG

VLDL secretion is a regulated process that depends on the availability of lipids, apoB and MTP. Our aim was to investigate the effect of liver denervation upon the secretion of VLDL and the expression of proteins involved in this process. Denervation was achieved by applying a 85% phenol solution onto the portal tract, while control animals were treated with 9% NaCl. VLDL secretion was evaluated by the Tyloxapol method. The hepatic concentration of TAG and cholesterol, and the plasma concentration of TAG, cholesterol, VLDL-TAG, VLDL-cholesterol and HDL-cholesterol were measured, as well as mRNA expression of proteins involved in the process of VLDL assembly. Hepatic acinar distribution of MTP and apoB was evaluated by immunohistochemistry. Denervation increased plasma concentration of cholesterol (125.3 +/- 10.1 vs. 67.1 +/- 4.9 mg dL(-1)) and VLDL-cholesterol (61.6 +/- 5.6 vs. 29.4 +/- 3.3 mg dL(-1)), but HDL-cholesterol was unchanged (45.5 +/- 6.1 vs. 36.9 +/- 3.9 mg dL(-1)). Secretion of VLDL-TAG (47.5 +/- 23.8 vs. 148.5 +/- 27.4 mg dL h(-1)) and mRNA expression of CPT I and apoB were reduced (p < 0.01) in the denervated animals. MTP and apoB acinar distribution was not altered in the denervated animals, but the intensity of the reaction was reduced in relation to controls.     

Investigation of the hepatic respiration and liver zonation on rat hepatocytes using an integrated oxygen biosensor in a microscale device

The development of an in vitro functional liver zonation model is a major issue to reproduce physiological liver features. Oxygen concentration is one of the potential explanations of a primary regulating factor of zonation. In this frame, we investigated the oxygen gradient inside a microfluidic device containing rat hepatocyte cultures. The device integrated a platinum (Pt) (II) octaethylporphyrin sensor, allowing a 2D mapping of the oxygen concentration. After 3 hr adhesion of the hepatocytes, the sensor indicated an intense oxygen depletion, leading to an oxygen shortage in the center of the device. After a 30 min perfusion of the culture medium, we monitored the formation of the oxygen gradient along the culture due to cellular respiration. The profile of the oxygen gradient was modulated and controlled by increasing either the perfusion flow rate or the device thickness. In addition, the oxygen gradient was time dependent as far as it decreased with the time of culture. Perivenous and periportal liver patterns were characterized by the immunostaining of the hepatic markers. We put in evidence a spatio temporal hepatic organization. We observed the overexpression since 24 hr of perfusion of the APC and PCK1 proteins upstream in the oxygen-rich area of the device. The overexpression of GS, GCK, CYP1A, and HIFα proteins were observed downstream in the oxygen-poor area. Then, CYP3A2 and β-catenin spatial reorganization was achieved after 48 hr of culture. The results presented a partial zonation-like pattern that was superimposed with an oxygen gradient profile.     

High liver lipoprotein lipase activity in hyperlipemic developing rats from undernourished pregnant mothers

To study the potential relationship between circulating triacylglycerol (TAG) levels and lipoprotein lipase (LPL) activity in the newborn rat liver, pups from undernourished or normal control mothers were nursed by normal dams, and studied at 0, 1, 15 or 30 days of age. Plasma TAG levels and liver TAG concentration increased more in pups from undernourished mothers than they did in controls. At birth, liver LPL activity was similarly high in both groups but, whereas in controls it decreased progressively after birth, in pups from undernourished mothers it remained stable until 15 days of age. Results suggest that the hypertriglyceridemia present in pups from undernourished mothers may be responsible for the sustained high LPL activity in their liver which may enhance the hepatic uptake of circulating TAG.     

不同灌注方法对原位肝移植麻醉的临床效果分析

目的:观察下腔静脉逆灌注法与常规门静脉正向灌注法应用于经典非转流原位肝移植手术的临床效果,并比较两种灌注法对新肝期再灌注后综合征的影响。方法:回顾性分析我院行经典非转流肝移植手术的患者60例,按照灌注方法的不同分为两组:A组(30例)采用下腔静脉逆灌注法;B组(30例)采用门静脉正向灌注法。比较两组患者围手术期的血流动力学指标、体温、内环境的改变等情况。结果:①分别在新肝开放即刻、新肝期5分钟及15分钟观察两组患者的平均动脉压、肺动脉压楔压、心率变化程度及心脏指数,A组均小于B组,差异有统计学意义(P0.05);②分别于新肝期5分钟及15分钟观察两组患者的中心静脉压,A组低于B组(P0.01);③观察开放时两组患者的最低温度,A组高于B组(P0.01);④观察血管活性药物用量,A组少于B组,差异有统计学意义(P0.05)。结论:经典非转流肝移植术中,采取逆灌注方法可减少术后再灌注综合征的发生。两种灌注方式均需要加强对血流动力学、体温、酸碱平衡的监测与调整,做到针对性的麻醉管理。     

Liver parenchyma heterogeneity in the response to extracellular NAD+

The perfused rat liver responds intensely to NAD+ infusion (20-100 microM). Increases in portal perfusion pressure and glycogenolysis and transient inhibition of oxygen consumption are some of the effects that were observed. The aim of the present work was to investigate the distribution of the response to extracellular NAD+ along the hepatic acinus. The bivascularly perfused rat liver was used. Various combinations of perfusion directions (antegrade and retrograde) and infusion routes (portal vein, hepatic vein and hepatic artery) were used in order to supply NAD+ to different regions of the liver parenchyma, also taking advantage of the fact that its extracellular transformation generates steep concentration gradients. Oxygen uptake was stimulated by NAD+ in retrograde perfusion (irrespective of the infusion route) and transiently inhibited in antegrade perfusion. This indicates that the signal causing oxygen uptake inhibition is generated in the periportal area. The signal responsible for oxygen uptake stimulation is homogenously distributed. Stimulation of glucose release was more intense when NAD+ was infused into the portal vein or into the hepatic artery, indicating that stimulation of glycogenolysis predominates in the periportal area. The increases in perfusion pressure were more pronounced when the periportal area was supplied with NAD+ suggesting that the vasoconstrictive elements responding to NAD+ predominate in this region. The response to extracellular NAD+ is thus unequally distributed in the liver. As a paracrine agent, NAD+ is likely to be released locally. It can be concluded that its effects will be different depending on the area where it is released.     

The action of extracellular NAD+ on gluconeogenesis in the perfused rat liver

In the rat liver NAD⁺ infusion produces increases in portal perfusion pressure and glycogenolysis and transient inhibition of oxygen consumption. The aim of the present work was to investigate the possible action of this agent on gluconeogenesis using lactate as a gluconeogenic precursor. Hemoglobin-free rat liver perfusion in antegrade and retrograde modes was used with enzymatic determination of glucose production and polarographic assay of oxygen uptake. NAD⁺ infusion into the portal vein (antegrade perfusion) produced a concentration-dependent (25–100 μM) transient inhibition of oxygen uptake and gluconeogenesis. For both parameters inhibition was followed by stimulation. NAD⁺ infusion into the hepatic vein (retrograde perfusion) produced only transient stimulations. During Ca²⁺-free perfusion the action of NAD⁺ was restricted to small transient stimulations. Inhibitors of eicosanoid synthesis with different specificities (indo-methacin, nordihydroguaiaretic acid, bromophenacyl bromide) either inhibited or changed the action of NAD⁺. The action of NAD⁺ on gluconeogenesis is probably mediated by eicosanoids synthesized in non-parenchymal cells. As in the fed state, in the fasted condition extracellular NAD⁺ is also able to exert two opposite effects, inhibition and stimulation. Since inhibition did not manifest significantly in retrograde perfusion it is likely that the generating signal is located in pre-sinusoidal regions.     

Technique of Subnormothermic Ex Vivo Liver Perfusion for the Storage,Assessment, and Repair of Marginal Liver Grafts

The success of liver transplantation has resulted in a dramatic organ shortage. In most transplant regions 20-30% of patients on the waiting list for liver transplantation die without receiving an organ transplant or are delisted for disease progression. One strategy to increase the donor pool is the utilization of marginal grafts, such as fatty livers, grafts from older donors, or donation after cardiac death (DCD). The current preservation technique of cold static storage is only poorly tolerated by marginal livers resulting in significant organ damage. In addition, cold static organ storage does not allow graft assessment or repair prior to transplantation.These shortcomings of cold static preservation have triggered an interest in warm perfused organ preservation to reduce cold ischemic injury, assess liver grafts during preservation, and explore the opportunity to repair marginal livers prior to transplantation. The optimal pressure and flow conditions, perfusion temperature, composition of the perfusion solution and the need for an oxygen carrier has been controversial in the past.In spite of promising results in several animal studies, the complexity and the costs have prevented a broader clinical application so far. Recently, with enhanced technology and a better understanding of liver physiology during ex vivo perfusion the outcome of warm liver perfusion has improved and consistently good results can be achieved.This paper will provide information about liver retrieval, storage techniques, and isolated liver perfusion in pigs. We will illustrate a) the requirements to ensure sufficient oxygen supply to the organ, b) technical considerations about the perfusion machine and the perfusion solution, and c) biochemical aspects of isolated organs.     

Bivascular liver perfusion in the anterograde and retrograde modes: Zonation of the response to inhibitors of oxidative phosphorylation

The action of cyanide (500 μM ), 2,4-dinitrophenol (50 μM ) and atractyloside (100 μM ) on glycogen catabolism and oxygen uptake was investigated in the bivascularly perfused liver of fed rats. Cyanide, 2,4-dinitrophenol and atractyloside were infused at identical rates into the hepatic artery in either the anterograde or retrograde perfusion. The accessible aqueous cell spaces were determined by means of the multiple-indicator dilution technique. Glucose release, oxygen uptake and glycolysis were measured as metabolic parameters. Oxygen uptake changes per unit cell space caused by atractyloside (inhibition) and 2,4-dinitrophenol (stimulation) were equal in the retrograde perfusion (periportal cells) and the anterograde perfusion (space enriched in perivenous cells); the decreases caused by cyanide were higher in the retrograde perfusion. Glucose release from periportal cells was not increased upon inhibition of oxidative phosphorylation, a phenomenon which was independent of the mechanism of action of the inhibitor. There were nearly identical changes in glycolysis in the periportal and perivenous cells. It was concluded that: (1) oxygen concentration in the perfused rat liver, if maintained above 100 μM , had little influence on the zonation of the respiratory activity; (2) in spite of the lower activities of the key enzymes of glycolysis in the periportal hepatocytes, as assayed under standard conditions, these cells were as effective as the perivenous ones in generating ATP in the cytosol when oxidative phosphorylation was impaired; (3) the key enzymes of glycogenolysis and glycolysis in periportal and perivenous cells responded differently to changes in the energy charge.     

Apolipoprotein B and triacylglycerol secretion in human triacylglycerol hydrolase transgenic mice

Apolipoprotein B (apoB)-containing lipoproteins play a critical role in whole body lipid homeostasis and the pathogenesis of atherosclerosis. The assembly of hepatic apoB-containing lipoproteins, VLDL, is governed by the availability of lipids, including triacylglycerol (TG). The majority of TG associated with VLDL is derived from the hepatic cytoplasmic lipid stores by a process involving lipolysis followed by reesterification. Microsomal triacylglycerol hydrolase (TGH) has been demonstrated to play a role in the lipolysis/reesterification process. To evaluate the potential regulatory role of TGH in hepatic VLDL assembly, we developed inducible transgenic mice expressing a human TGH minigene under the control of the mouse metallothionein promoter. Induction of human TGH by zinc resulted in liver-specific expression of the enzyme associated with 3- to 4-fold increases in lipolytic activity that could be attenuated with a TGH-specific inhibitor. Augmented TGH activity led to increased secretion of newly synthesized apoB and plasma TG levels. These results suggest that increased hepatic expression of TGH leads to a more proatherogenic plasma lipid and apoB profile.     

Hepatic heterogeneity in the response to ATP studied in the bivascularly perfused rat liver

The zonation of the purinergic action of ATP in the hepatic parenchyma was investigated in the bivascularly perfused rat liver by means of anterograde and retrograde perfusion. Livers from fed rats were used, and ATP was infused according to four different experimental protocols: (A) anterograde perfusion and ATP infusion via the portal vein; (B) anterograde perfusion and ATP via the hepatic artery; (C) retrograde perfusion and ATP via the hepatic vein; (D) retrograde perfusion and ATP via the hepatic artery. The following metabolic parameters were measured: glucose release, lactate production and oxygen consumption. The hemodynamic effects were evaluated by measuring the sinusoidal mean transit times by means of the indicator-dilution technique. ATP was infused during 20 min at four different rates (between 0.06-0.77 µmol min_-1 g liver_-1; 20-200 µM) in each of the four experimental protocols.The results that were obtained allow several conclusions with respect to the localization of the effects of ATP along the hepatic acini: (1) In retrograde perfusion the sinusoidal mean transit times were approximately twice those observed in anterograde perfusion. ATP increased the sinusoidal mean transit times only in retrograde perfusion (protocols C and D). The effect was more pronounced with protocol D. These results allow the conclusion that the responsive vasoconstrictive elements are localized in a pre-sinusoidal region; (2) All hepatic cells, periportal as well as perivenous, were able to metabolize ATP, so that concentration gradients were generated with all experimental protocols. Extraction of ATP was more pronounced in retrograde perfusion, an observation that can be attributed, partly at least, to the longer sinusoidal transit times. In anterograde perfusion, the extraction of ATP was time-dependent, a phenomenon that cannot be satisfactorily explained with the available data; (3) ATP produced a transient initial inhibition of oxygen uptake when protocols A and B were employed. These protocols are the only ones in which the cells situated shortly after the intrasinusoidal confluence of the portal vein and the hepatic artery were effectively supplied with ATP. The decrease in oxygen consumption was more pronounced at low ATP infusions when protocol B was employed. These observations allow the conclusion that the former phenomenon is localized mainly in cells situated shortly after the intrasinusoidal confluence of the portal vein and hepatic artery. Oxygen consumption in all other cells, especially the proximal periportal ones, is increased by ATP; (4) In agreement with previous data found in the literature, glycogenolysis stimulation by ATP was more pronounced in the periportal region. The cells that respond more intensively are not the proximal periportal ones, but those situated in the region of the intrasinusoidal confluence of the portal vein and the hepatic artery.     

Perfusion flow rate substantially contributes to the performance of the HepaRG‐AMC‐bioartificial liver

Bioartificial livers (BALs) are bioreactors containing liver cells that provide extracorporeal liver support to liver‐failure patients. Theoretically, the plasma perfusion flow rate through a BAL is an important determinant of its functionality. Low flow rates can limit functionality due to limited substrate availability, and high flow rates can induce cell damage. This hypothesis was tested by perfusing the AMC‐BAL loaded with the liver cell line HepaRG at four different medium flow rates (0.3, 1.5, 5, and 10 mL/min). Hepatic functions ammonia elimination, urea production, lactate consumption, and 6β‐hydroxylation of testosterone showed 2–20‐fold higher rates at 5 mL/min compared to 0.3 mL/min, while cell damage remained stable. However, at 10 mL/min cell damage was twofold higher, and maximal hepatic functionality was not changed, except for an increase in lactate elimination. On the other hand, only a low flow rate of 0.3 mL/min allowed for an accurate measurement of the ammonia and lactate mass balance across the bioreactor, which is useful for monitoring the BAL's condition during treatment. These results show that (1) the functionality of a BAL highly depends on the perfusion rate; (2) there is a universal optimal flow rate based on various function and cell damage parameters (5 mL/min for HepaRG‐BAL); and (3) in the current set‐up the mass balance of substrate, metabolite, or cell damage markers between in‐and out‐flow of the bioreactor can only be determined at a suboptimal, low, perfusion rate (0.3 mL/min for HepaRG‐BAL). Biotechnol. Bioeng. 2012; 109: 3182–3188. © 2012 Wiley Periodicals, Inc.