Induction and progression of cholangiofibrosis in rat liver injured by oral administration of furan.

Cholangiofibrosis is a structural anomaly that precedes the development of cholangiocarcinoma in some rodent models. In this article, the authors examine the contribution of the epithelial and mesenchymal cells in the pathogenesis of this complex lesion. Furan was administered to rats by gavage in corn oil at 30 mg/kg b.w. (five daily doses per week) and livers were sampled between eight hr to three months. Characteristically the administration of furan caused centrilobular injury, and restoration was accomplished by proliferation of hepatocytes. Some areas of the liver were, however, more severely affected, and here, injury extended into portal and capsular areas, which resulted in a rapid proliferation of ductular cells that extended into the parenchyma accompanied by a subtype of liver fibroblasts. These ductules either differentiated into hepatocytes, with loss of the associated fibroblasts, or progressed to form tortuous ductular structures that replaced much of the parenchyma, leading to cholangiofibrosis. Although it is unclear what determines the difference in the hepatic response, a loss of micro-environmental cues that instigate hepatocyte differentiation and termination of the hepatocyte stem cell repair response may be perturbed by continual furan administration that results in an irreversible expansile lesion that may mimic the features of cholangiocarcinoma.

An introduction to QT interval prolongation and non-clinical approaches to assessing and reducing risk.

Owing to its association with Torsades de Pointes, drug-induced QT interval prolongation has been and remains a significant hurdle to the development of safe, effective medicines. Genetic and pharmacological evidence highlighting the pivotal role the human ether-a-go-go-related gene (hERG) channel was a critical step in understanding how to start addressing this issue. It led to the development of hERG assays with the rapid throughput needed for the short timescales required in early drug discovery. The resulting volume of hERG data has fostered in silico models to help chemists design compounds with reduced hERG potency. In early drug discovery, a pragmatic approach based on exceeding a given potency value has been required to decide when a compound is likely to carry a low QT risk, to support its progression to late-stage discovery. At this point, the in vivo efficacy and metabolism characteristics of the potential drug are generally defined, as well its safety profile, which includes usually a dog study to assess QT interval prolongation risk. The hERG and in vivo QT data, combined with the likely indication and the estimated free drug level for efficacy, are put together to assess the risk that the potential drug will prolong QT in man. Further data may be required to refine the risk assessment before making the major investment decisions for full development. The non-clinical data are essential to inform decisions about compound progression and to optimize the design of clinical QT studies.

Evidence of oxidative stress and associated DNA damage, increased proliferative drive, and altered gene expression in rat liver produced by the cholangiocarcinogenic agent furan.

Furan is a potent cholangiocarcinogen in rat by an as yet undefined mechanism. The risk to man remains unclear. Using a time-course stop study design, we have investigated the potential of furan to induce oxidative stress and DNA damage associated with inflammatory and regenerative responses in rat liver. Furan was administered via oral gavage (30 mg/kg b.w. 5 daily doses per week), and livers were analyzed at time points between eight hr and three months. A one-month recovery group previously treated for three months was also included. There was a marked association between CYP2E1 expression and DNA oxidation (8-oxo-dG) in areas of centrilobular hepatocyte necrosis seen after a single dose. After one-month recovery from three-month treatment, 8-oxo-dG was still observed in areas of furan-induced cholangiofibrosis. Furan-induced changes in the expression of various genes associated with oxidative stress, DNA damage, and cell cycle control were identified during treatment and recovery. We propose that furan-induced cholangiocarcinomas emerge from areas of cholangiofibrosis as a result of a combination of chronic, persistent indirect damage to DNA through oxygen radicals coupled with persistent proliferative signals, including loss of connexin 32, that act to convert this DNA damage to fixed mutations.

In vitro models of proarrhythmia.

Proarrhythmia models use electrophysiological markers to predict the risk of torsade de pointes (TdP) in patients. The set of variables used by each model to predict the torsadogenic propensity of a drug has been reported to correlate with clinical outcome; however, these reports should be interpreted cautiously as no model has been independently assessed. Each model is discussed along with its merits and shortcomings; none, as yet, having shown a predictive value that makes it clearly superior to the others. As predictive as these models may become, extrapolation of results directly to the clinic must be exercised with caution. The use of in silico models, from subcellular to whole system, is rapidly beginning to form the first line of screening activity in many drug discovery programmes, indicating that biological experimentation may become secondary to analysis by simulation. In vitro proarrhythmia models challenge current perceptions of appropriate surrogates for TdP in man and question existing non-clinical strategies for assessing proarrhythmic risk. The rapid emergence of such models, compounded by the lack of a clear understanding of the key proarrhythmic mechanisms has resulted in a regulatory reluctance to embrace such models. The wider acceptance of proarrhythmia models is likely to occur when there is a clear understanding and agreement on the key proarrhythmia mechanisms. With greater acceptance and ongoing improvements, these models have the potential to unravel the complex mechanisms underlying TdP.

Strategies to reduce the risk of drug-induced QT interval prolongation: a pharmaceutical company perspective.

Drug-induced prolongation of the QT interval is having a significant impact on the ability of the pharmaceutical industry to develop new drugs. The development implications for a compound causing a significant effect in the 'Thorough QT/QTc Study' -- as defined in the clinical regulatory guidance (ICH E14) -- are substantial. In view of this, and the fact that QT interval prolongation is linked to direct inhibition of the hERG channel, in the early stages of drug discovery the focus is on testing for and screening out hERG activity. This has led to understanding of how to produce low potency hERG blockers whilst retaining desirable properties. Despite this, a number of factors mean that when an integrated risk assessment is generated towards the end of the discovery phase (by conducting at least an in vivo QT assessment) a QT interval prolongation risk is still often apparent; inhibition of hERG channel trafficking and partitioning into cardiac tissue are just two confounding factors. However, emerging information suggests that hERG safety margins have high predictive value and that when hERG and in vivo non-clinical data are combined, their predictive value to man, whilst not perfect, is >80%. Although understanding the anomalies is important and is being addressed, of greater importance is developing a better understanding of TdP, with the aim of being able to predict TdP rather than using an imperfect surrogate marker (QT interval prolongation). Without an understanding of how to predict TdP risk, high-benefit drugs for serious indications may never be marketed.

Effect of chronic MK-801 and/or phenytoin on the acquisition of complex behaviors in rats.

The purpose of the present experiment was to assess the effects of chronic MK-801 (an N-methyl-D-aspartate receptor antagonist) and/or phenytoin (a sodium channel blocker) treatment on behavioral acquisition and performance in rats. Learning, audio/visual discrimination and motivation were modeled using incremental repeated acquisition (IRA), audio/visual discrimination (AVD) and progressive ratio (PR) tasks, respectively. MK-801 and/or phenytoin were administered daily, 7 days/week by orogastric gavage beginning just after weaning on postnatal day (PND) 23 and continuing until PND 306. Monday through Friday behavioral assessments began on PND 27 and continued until PND 430. Throughout treatment, subjects in the high dose MK-801 (1.0 mg/kg/day) and the high dose drug combination (1.0 mg/kg/day MK-801+150 mg/kg/day phenytoin) groups exhibited decreased body weight gains compared to control subjects. For these two affected groups, response rates were also decreased in all tasks. Task acquisition, as evidenced by an increase in response accuracy, was decreased for both these groups in the AVD task, but only for the high dose MK-801 group in the IRA task. The data suggest that chronic MK-801 treatment adversely affects the acquisition of IRA and AVD task performance and that the inclusion of phenytoin in the MK-801 dosing regimen blocks some of the adverse effects of chronic MK-801 treatment on IRA task acquisition. These findings are in marked contrast with those observed in nonhuman primates and suggest important species differences associated with chronic exposure to compounds that block NMDA receptors and/or sodium channels.

Behavioral effects associated with chronic ketamine or remacemide exposure in rats.

The effects of chronic exposure to ketamine or remacemide on the acquisition and performance of food-reinforced operant behaviors was assessed in female Sprague-Dawley rats. Ketamine is an anesthetic N-methyl-D-aspartate (NMDA) receptor antagonist, whereas remacemide is an active central nervous system compound with both NMDA receptor antagonist and sodium channel blocking properties. Learning, audio/visual discrimination and motivation were modeled using incremental repeated acquisition (IRA), audio/visual discrimination (AVD) and progressive ratio (PR) tasks, respectively. Ketamine (10 or 100 mg/kg/day), remacemide (100 or 150 mg/kg/day) or water was administered daily (7 days/week) via orogastric gavage beginning on postnatal day (PND) 23 and continuing until PND 257. Monday through Friday behavioral assessments began on PND 27 and continued until PND 383. Chronic treatment with the high dose of ketamine decreased response rate in all tasks suggesting decreased motivation or motoric capabilities. Chronic treatment with ketamine or remacemide had no effect on the acquisition of IRA task performance at any dose tested. While chronic treatment with either high-dose ketamine or low-dose remacemide only delayed the acquisition of AVD task performance for a brief period midway through treatment, chronic treatment with high-dose remacemide delayed the acquisition of AVD task performance until late in treatment. The findings for ketamine are quite different from those of MK-801 (the prototypic NMDA receptor antagonist) in a previous rat study in which MK-801 severely disrupted the acquisition of both IRA and AVD task performances. These observations suggest important differences in the mechanism of action between ketamine and MK-801. For example, ketamine has a much lower binding affinity than MK-801 for the NMDA receptor, the dopamine transporter and the dopamine D2 receptor. In addition, the findings for remacemide observed in rats are in marked contrast with those seen in monkeys where chronic remacemide had profound disruptive effects on the acquisition of both IRA and AVD task performances and suggest important species differences.

A rabbit Langendorff heart proarrhythmia model: predictive value for clinical identification of Torsades de Pointes.

BACKGROUND AND PURPOSE: The rabbit isolated Langendorff heart model (SCREENIT) was used to investigate the proarrhythmic potential of a range of marketed drugs or drugs intended for market. These data were used to validate the SCREENIT model against clinical outcomes. EXPERIMENTAL APPROACH: Fifty-five drugs, 3 replicates and 2 controls were tested in a blinded manner. Proarrhythmia variables included a 10% change in MAPD(60), triangulation, instability and reverse frequency-dependence of the MAP. Early after-depolarisations, ventricular tachycardia, TdP and ventricular fibrillation were noted. Data are reported at nominal concentrations relative to EFTPC(max). Proarrhythmic scores were assigned to each drug and each drug category. KEY RESULTS: Category 1 and 2 drugs have the highest number of proarrhythmia variables and overt proarrhythmia while Category 5 drugs have the lowest, at every margin. At 30-fold the EFTPC(max), the mean proarrhythmic scores are: Category 1, 101+/-24; Category 2, 101+/-14; Category 3, 72+/-20; Category 4, 59+/-16 and Category 5, 22+/-9 points. Only drugs in Category 5 have mean proarrhythmic scores, below 30-fold, that remain within the Safety Zone. CONCLUSIONS AND IMPLICATIONS: A 30-fold margin between effects and EFTPC(max) is sufficiently stringent to provide confidence to proceed with a new chemical entity, without incurring the risk of eliminating potentially beneficial drugs. The model is particularly useful where compounds have small margins between the hERG IC(50) and predicted EFTPC(max). These data suggest this is a robust and reliable assay that can add value to an integrated QT/TdP risk assessment.

Optimisation and validation of a medium-throughput electrophysiology-based hERG assay using IonWorks HT.

INTRODUCTION: Regulatory and competitive pressure to reduce the QT interval prolongation risk of potential new drugs has led to focus on methods to test for inhibition of the human ether-a-go-go-related gene (hERG)-encoded K+ channel, the primary molecular target underlying this safety issue. Here we describe the validation of a method that combines medium-throughput with direct assessment of channel function. METHODS: The electrophysiological and pharmacological properties of hERG were compared using two methods: conventional, low-throughput electrophysiology and planar-array-based, medium-throughput electrophysiology (IonWorks HT). A pharmacological comparison was also made between IonWorks HT and an indirect assay (Rb+ efflux). RESULTS: Basic electrophysiological properties of hERG were similar whether recorded conventionally (HEK cells) or using IonWorks HT (CHO cells): for example, tail current V1/2 -12.1+/-5.0 mV (32) for conventional and -9.5+/-6.0 mV (46) for IonWorks HT (mean+/-S.D. (n)). A key finding was that as the number of cells per well was increased in IonWorks HT, the potency reported for a given compound decreased. Using the lowest possible cell concentration (250,000 cells/ml) and 89 compounds spanning a broad potency range, the pIC50 values from IonWorks HT (CHO-hERG) were found to correlate well with those obtained using conventional methodology (HEK-hERG)(r=0.90; p<0.001). Further validation using CHO-hERG cells with both methods confirmed the correlation (r=0.94; p<0.001). In contrast, a comparison of IonWorks HT and Rb+ efflux data with 649 compounds using CHO-hERG cells showed that the indirect assay consistently reported compounds as being, on average, 6-fold less potent, though the differences varied depending on chemical series. DISCUSSION: The main finding of this work is that providing a relatively low cell concentration is used in IonWorks HT, the potency information generated correlates well with that determined using conventional electrophysiology. The effect on potency of increasing cell concentration may relate to a reduced free concentration of test compound owing to partitioning into cell membranes. In summary, the IonWorks HT hERG assay can generate pIC50 values based on a direct assessment of channel function in a timeframe short enough to influence chemical design.

Use of in vitro methods to predict QT prolongation.

The inhibition of the hERG-encoded potassium channel can lead to prolongation of the cardiac action potential--manifested as a prolongation of the QT interval on the ECG. Although QT interval prolongation is not dangerous per se, in a small percentage of cases, it is associated with a potentially fatal arrhythmia: Torsades de Pointes (TdP). This channel type is pharmacologically promiscuous, so many compounds have caused QT interval prolongation in man and this has led to drugs being withdrawn from the market following evidence of TdP. From a drug discovery perspective, focusing as early as possible on screening out hERG activity is important. Retrospective analysis of hERG potency versus clinical incidence of TdP suggests provisional safety margins that could be used as target values by medicinal chemists. Large safety margins will not always be possible; however, and in such circumstances, if the risk-benefit ratio still favours developing the compound, a pre-clinical assessment of the likelihood that any QT interval prolongation will or will not lead to TdP in man may be important. An isolated rabbit heart model of arrhythmia shows promise in this respect, based on a comparison of clinical data with that obtained from this assay. Specific regulatory guidance on this topic is still in the draft form but the pre-clinical document (ICH S7B) contains a largely useful perspective on how an integrated risk assessment could be formed using in vitro and in vivo assays. The role of this document is evolving however, since the draft clinical guideline (E14) suggests that irrespective of the pre-clinical data, a thorough clinical ECG study will be required at some point during development.

Relationships between preclinical cardiac electrophysiology, clinical QT interval prolongation and torsade de pointes for a broad range of drugs: evidence for a provisional safety margin in drug development.

OBJECTIVE: To attempt to determine the relative value of preclinical cardiac electrophysiology data (in vitro and in vivo) for predicting risk of torsade de pointes (TdP) in clinical use. METHODS: Published data on hERG (or I(Kr)) activity, cardiac action potential duration (at 90% repolarisation; APD(90)), and QT prolongation in dogs were compared against QT effects and reports of TdP in humans for 100 drugs. These data were set against the free plasma concentrations attained during clinical use (effective therapeutic plasma concentrations; ETPC(unbound)). The drugs were divided into five categories: (1) Class Ia and III antiarrhythmics; (2) Withdrawn from market due to TdP; (3) Measurable incidence/numerous reports of TdP in humans; (4) Isolated reports of TdP in humans; (5) No reports of TdP in humans. RESULTS: Data from hERG (or I(Kr)) assays in addition to ETPC(unbound) data were available for 52 drugs. For Category 1 drugs, data for hERG/I(Kr) IC(50), APD(90), QTc in animals and QTc in humans were generally close to or superimposed on the ETPC(unbound) values. This relationship was uncoupled in the other categories, with more complex relationships between the data. In Category 1 (except amiodarone), the ratios between hERG/I(Kr) IC(50) and ETPC(unbound) (max) ranged from 0.1- to 31-fold. Similar ranges were obtained for drugs in Category 2 (0.31- to 13-fold) and Category 3 (0.03- to 35-fold). A large spread was found for Category 4 drugs (0.13- to 35700-fold); this category embraced an assortment of mechanisms ranging from drugs which may well be affecting I(Kr) currents in clinical use (e.g. sparfloxacin) to others such as nifedipine (35700-fold) where channel block is not involved. Finally, for the majority of Category 5 drugs there was a >30-fold separation between hERG/I(Kr) activity and ETPC(unbound) values, with the notable exception of verapamil (1.7-fold), which is free from QT prolongation in man; this is probably explained by its multiple interactions with cardiac ion channels. CONCLUSIONS: The dataset confirms the widely-held belief that most drugs associated with TdP in humans are also associated with hERG K(+) channel block at concentrations close to or superimposed upon the free plasma concentrations found in clinical use. A 30-fold margin between C(max) and hERG IC(50) may suffice for drugs currently undergoing clinical evaluation, but for future drug discovery programmes, pharmaceutical companies should consider increasing this margin, particularly for drugs aimed at non-debilitating diseases. However, interactions with multiple cardiac ion channels can either mitigate or exacerbate the prolongation of APD and QT that would ensue from block of I(Kr) currents alone, and delay of repolarisation per se is not necessarily torsadogenic. Clearly, an integrated assessment of in vitro and in vivo data is required in order to predict the torsadogenic risk of a new candidate drug in humans.

Assessing the potential toxicity of MK-801 and remacemide: chronic exposure in juvenile rhesus monkeys.

The present experiment examined the effects of chronic exposure to either 0.1 or 1.0 mg/kg MK-801 [a selective N-methyl-D-aspartate (NMDA) receptor antagonist] or 20.0 or 50.0 mg/kg remacemide (an NMDA receptor antagonist which also blocks fast sodium channels) in juvenile rhesus monkeys. Endpoints were monitored to provide a general index of subjects' health and included measures of clinical chemistry, hematology, ophthalmology, spontaneous home-cage behavior, and peak drug plasma levels. In general, both drugs were well tolerated and produced no treatment-related effects during 2 years of dosing and assessment. Periodic plasma drug level determinations provided limited evidence that both compounds may induce their own metabolism. The present results contrast sharply with previously reported effects of long-lasting impairments in the acquisition of incremental learning and in the development of color and position discrimination in these same subjects. These observations highlight the importance of collecting a broad range of toxicology data, including tests of cognitive function, to make comprehensive assessments of new drug safety. In the present case, the less obvious effects of these drugs on cognition defined the toxicologic response.

LPS-sensory peptide communication in experimental cystitis.

Stimulation of sensory nerves can lead to release of peptides such as substance P (SP) and consequently to neurogenic inflammation. We studied the role of bacterial lipopolysaccharide (LPS) in regulating SP-induced inflammation. Experimental cystitis was induced in female mice by intravesical instillation of SP, LPS, or fluorescein-labeled LPS. Uptake of fluorescein-labeled LPS was determined by confocal analysis, and bladder inflammation was determined by morphological analysis. SP was infused into the bladders of some mice 24 h after exposure to LPS. In vitro studies determined the capacity of LPS and SP to induce histamine and cytokine release by the bladder. LPS was taken up by urothelial cells and distributed systemically. Twenty-four hours after instillation of LPS or SP, bladder inflammation was characterized by edema and leukocytic infiltration of the bladder wall. LPS pretreatment enhanced neutrophil infiltration induced by SP, increased in vitro release of histamine, tumor necrosis factor-alpha, and interferon-gamma, and significantly reduced transforming growth factor-beta1 release. These findings suggest that LPS amplifies neurogenic inflammation, thereby playing a role in the pathogenesis of neurogenic cystitis.

AQP2 is a substrate for endogenous PP2B activity within an inner medullary AKAP-signaling complex.

We have demonstrated that inner medullary collecting duct (IMCD) heavy endosomes purified from rat kidney IMCD contain the type II protein kinase A (PKA) regulatory subunit (RII), protein phosphatase (PP)2B, PKCzeta, and an RII-binding protein (relative molecular mass ~90 kDa) representing a putative A kinase anchoring protein (AKAP). Affinity chromatography of detergent-solubilized endosomes on cAMP-agarose permits recovery of a protein complex consisting of the 90-kDa AKAP, RII, PP2B, and PKCzeta. With the use of small-particle flow cytometry, RII and PKCzeta were localized to an identical population of endosomes, suggesting that these proteins are components of an endosomal multiprotein complex. (32)P-labeled aquaporin-2 (AQP2) present in these PKA-phosphorylated endosomes was dephosphorylated in vitro by either addition of exogenous PP2B or by an endogenous endosomal phosphatase that was inhibited by the PP2B inhibitors EDTA and the cyclophilin-cyclosporin A complex. We conclude that IMCD heavy endosomes possess an AKAP multiprotein-signaling complex similar to that described previously in hippocampal neurons. This signaling complex potentially mediates the phosphorylation of AQP2 to regulate its trafficking into the IMCD apical membrane. In addition, the PP2B component of the AKAP-signaling complex could also dephosphorylate AQP2 in vivo.

Mast cell regulation of inflammation and gene expression during antigen-induced bladder inflammation in mice.

Mast cell numbers are significantly increased in bladder disorders including malignancy and interstitial cystitis, but their precise role has been difficult to determine. We characterized the role of mast cells on gene regulation associated with antigen-induced bladder inflammation in mice. For this purpose, we examined the responses in mast cell-deficient (Kit(W)/Kit(W-v)), congenic normal (+/+), and Kit(W)/Kit(W-v) mice that were reconstituted with bone marrow stem cells (BMR) to restore mast cells. All mice were actively sensitized and challenged intravesically with either saline or specific antigen. Bladder inflammation occurred in +/+ and BMR but not the Kit(W)/Kit(W-v) mice. Gene expression was determined using mouse cDNA expression arrays. Self-organizing maps, performed without preconditions, indicated gene expression changes dependent on the presence of mast cells. These genes were upregulated in bladders isolated from antigen challenge of +/+, not altered in Kit(W)/Kit(W-v), and were upregulated in BMR mice. Taken together these results demonstrate an important role for mast cells in allergic cystitis and indicate that mast cells can alter their environment by regulating tissue gene expression.

Differential effects of two NMDA receptor antagonists on cognitive-behavioral development in nonhuman primates I.

The present experiment examined effects of chronic exposure to remacemide (an N-methyl-D-aspartate [NMDA] antagonist which also blocks fast sodium channels) or MK-801 (which blocks NMDA receptors, exclusively) on learning and motivation in young rhesus monkeys. Remacemide (20 or 50 mg/kg/day) or MK-801 (0.1 or 1.0 mg/kg/day) was administered every day to separate groups of animals via orogastric gavage for up to 2 years. Immediately prior to dosing, 5 days per week (M--F), throughout the 2-year dosing period, an incremental repeated acquisition (IRA) task was used to assess learning and a progressive ratio (PR) task was used to assess motivation. The results indicate an effect of 50 mg/kg/day remacemide to impair learning (IRA) which persisted even after drug treatment was discontinued. MK-801 had no effect on learning but transiently increased motivation. Because the effects of remacemide occurred independently of changes in motivation or response rates, they are likely due to specific cognitive impairments and are not due to an inability of subjects to fulfill the motoric requirements of the task. The fact that MK-801 did not alter learning suggests that NMDA antagonism alone may be insufficient to produce learning deficits in young monkeys and that such deficits may rely on the ancillary blockade of fast sodium channels.

Differential effects of two NMDA receptor antagonists on cognitive--behavioral performance in young nonhuman primates II.

The present experiment examined the effects of chronic exposure to remacemide (an NMDA antagonist that also blocks fast sodium channels) or MK-801 (which blocks NMDA receptors more selectively) on the acquisition of color and position discrimination and short-term memory behavior in juvenile rhesus monkeys. Throughout the 2-year dosing period, a conditioned position responding (CPR) task was used to assess color and position discrimination and a delayed matching-to-sample (DMTS) task was used to assess memory. Chronic exposure to high doses of either drug delayed the acquisition of accurate color and position discrimination without altering response rates. In the case of MK-801, these effects abated within 6 months of the start of treatment. In the case of remacemide, the effects persisted for 17 months of dosing. Neither compound significantly altered performance of the short-term memory task at any time point or at any dose tested. The fact that the effects of remacemide on behavioral performance were more persistent than those seen for MK-801 suggests that tolerance may develop to the behavioral effects of MK-801, which does not develop to the effects of remacemide. Alternatively, these results may suggest that the concurrent antagonism of NMDA receptors and fast sodium channels may have more profound consequences for behavior than does the antagonism of NMDA receptors alone.

Optimized suspension culture: the rotating-wall vessel.

Suspension culture remains a popular modality, which manipulates mechanical culture conditions to maintain the specialized features of cultured cells. The rotating-wall vessel is a suspension culture vessel optimized to produce laminar flow and minimize the mechanical stresses on cell aggregates in culture. This review summarizes the engineering principles, which allow optimal suspension culture conditions to be established, and the boundary conditions, which limit this process. We suggest that to minimize mechanical damage and optimize differentiation of cultured cells, suspension culture should be performed in a solid-body rotation Couette-flow, zero-headspace culture vessel such as the rotating-wall vessel. This provides fluid dynamic operating principles characterized by 1) solid body rotation about a horizontal axis, characterized by colocalization of cells and aggregates of different sedimentation rates, optimally reduced fluid shear and turbulence, and three-dimensional spatial freedom; and 2) oxygenation by diffusion. Optimization of suspension culture is achieved by applying three tradeoffs. First, terminal velocity should be minimized by choosing microcarrier beads and culture media as close in density as possible. Next, rotation in the rotating-wall vessel induces both Coriolis and centrifugal forces, directly dependent on terminal velocity and minimized as terminal velocity is minimized. Last, mass transport of nutrients to a cell in suspension culture depends on both terminal velocity and diffusion of nutrients. In the transduction of mechanical culture conditions into cellular effects, several lines of evidence support a role for multiple molecular mechanisms. These include effects of shear stress, changes in cell cycle and cell death pathways, and upstream regulation of secondary messengers such as protein kinase C. The discipline of suspension culture needs a systematic analysis of the relationship between mechanical culture conditions and biological effects, emphasizing cellular processes important for the industrial production of biological pharmaceuticals and devices.