Maximizing the security of chaotic optical communications.

The practical application of chaotic optical communications has been limited by two aspects: the difficulty in concealing the time delay - a critical security parameter in feedback chaotic systems, and the difficulty of significantly enlarging the key space without complicating the implementation. Here we propose an architecture to break the above limits. By introducing a frequency-dependent group delay module with frequency tuning resolution of 1 MHz into the chaotic feedback loop, we demonstrate excellent time delay concealment effect, and an additional huge key space of 1048 can be achieved at the same time. The effectiveness is proved by both numerical simulation and experiment. Besides, the proposed scheme is compatible with the existing commercial optical communication systems, thus pave the way for high-speed secure optical communications.

Increased titer and reduced lactate accumulation in recombinant retrovirus production through the down-regulation of HIF1 and PDK.

Many mammalian cell lines used in the manufacturing of biopharmaceuticals exhibit high glycolytic flux predominantly channeled to the production of lactate. The accumulation of lactate in culture reduces cell viability and may also decrease product quality. In this work, we engineered a HEK 293 derived cell line producing a recombinant gene therapy retroviral vector, by down-regulating hypoxia inducible factor 1 (HIF1) and pyruvate dehydrogenase kinase (PDK). Specific productivity of infectious viral titers could be increased more than 20-fold for single gene knock-down (HIF1 or PDK) and more than 30-fold under combined down-regulation. Lactate production was reduced up to 4-fold. However, the reduction in lactate production, alone, was not sufficient to enhance the titer: high-titer clones also showed significant enrollment of metabolic routes not related to lactate production. Transcriptome analysis indicated activation of biological amines metabolism, detoxification routes, including glutathione metabolism, pentose phosphate pathway, glycogen biosynthesis and amino acid catabolism. The latter were validated by enzyme activity assays and metabolite profiling, respectively. High-titer clones also presented substantially increased transcript levels of the viral genes expression cassettes. The results herein presented demonstrate the impact of HIF1 and PDK down-regulation on the production performance of a mammalian cell line, reporting one of the highest fold-increase in specific productivity of infectious virus titers achieved by metabolic engineering. They additionally highlight the contribution of secondary pathways, beyond those related to lactate production, that can be also explored to pursue improved metabolic status favoring a high-producing phenotype.

Comparing maternal and perinatal outcomes in pregnancies complicated by preeclampsia superimposed chronic hypertension and preeclampsia alone.

AIM: The study was to determine whether preeclampsia with superimposed chronic hypertension results in worse maternal and perinatal outcomes than preeclampsia alone. MATERIALS AND METHODS: A retrospective study involving 850 pregnant women was conducted and divided into two groups: preeclampsia superimposed on chronic hypertension (group A, n = 84) and preeclampsia alone (group B, n = 766). The maternal and fetal outcomes of all subjects were collected and analyzed. RESULTS: There were no significant differences between the two groups in baseline information. However, the systolic and diastolic blood pressures in group A were significantly higher than those in group B (p < 0.05). The average interval between the onset of preeclampsia and the termination of pregnancy was significantly longer in group A as compared to group B. The incidence of serious maternal complications showed no differences between the two groups (p > 0.05). It showed a higher rate of neonatal respiratory distress syndrome and intracranial hemorrhage in group A than in group B (p < 0.05). CONCLUSIONS: Women in group A had higher risks of maternal and perinatal outcomes as compared to women in group B.

Three-dimensional multipotent progenitor cell aggregates for expansion, osteogenic differentiation and 'in vivo' tracing with AAV vector serotype 6.

Multipotent adult progenitor cells (MAPCs) are bone marrow-derived stem cells with a high growth rate suitable for therapeutical applications as three-dimensional (3D) aggregates. Combined applications of osteogenically differentiated MAPC (OD-MAPC) aggregates and adeno-associated viral vectors (AAV) in bone bioengineering are still deferred until information with regard to expansion technologies, osteogenic potential, and AAV cytotoxicity and transduction efficiency is better understood. In this study, we tested whether self-complementary AAV (scAAV) can potentially be used as a gene delivery system in an OD-MAPC-based 'in vivo' bone formation model in the craniofacial region. Both expansion of rat MAPC (rMAPC) and osteogenic differentiation with dexamethasone were also tested in 3D aggregate culture systems 'in vitro' and 'vivo'. rMAPCs grew as undifferentiated aggregates for 4 days, with a population doubling time of 37 h. After expansion, constant levels of Oct4 transcripts, and Oct4 and CD31 surface markers were observed, which constitute a hallmark of undifferentiated stage of rMAPCs. Dexamethasone effectively mediated rMAPC osteogenic differentiation by inducing the formation of a mineralized collagen type I network, and facilitated the activation of the wnt/ß-catenin, a crucial pathway in skeletal development. To investigate the genetic modification of rMAPCs grown as 3D aggregates before implantation, scAAV serotypes 2, 3 and 6 were evaluated. scAAV6 packaged with the enhanced green fluorescent protein expression cassette efficiently mediated long-term transduction (10 days) 'in vitro' and 'vivo'. The reporter transduction event allowed the tracing of OD-rMAPC (induced by dexamethasone) aggregates following OD-rMAPC transfer into a macro-porous hydroxyapatite scaffold implanted in a rat calvaria model. Furthermore, the scAAV6-transduced OD-rMAPCs generated a bone-like matrix with a collagenous matrix rich in bone-specific proteins (osteocalcin and osteopontin) in the scaffold macro-pores 10 days post-implantation. Newly formed bone was also observed in the interface between native bone and scaffold. The collective work supports future bone tissue engineering applications of 3D MAPC cultures for expansion, bone formation and the ability to alter genetically these cells using scAAV vectors.

Metabolic pathways recruited in the production of a recombinant enveloped virus: mining targets for process and cell engineering.

Biopharmaceuticals derived from enveloped virus comprise an expanding market of vaccines, oncolytic vectors and gene therapy products. Thus, increased attention is given to the development of robust high-titer cell hosts for their manufacture. However, the knowledge on the physiological constraints modulating virus production is still scarce and the use of integrated strategies to improve hosts productivity and upstream bioprocess an under-explored territory. In this work, we conducted a functional genomics study, including the transcriptional profiling and central carbon metabolism analysis, following the metabolic changes in the transition 'parental-to-producer' of two human cell lines producing recombinant retrovirus. Results were gathered into three comprehensive metabolic maps, providing a broad and integrated overview of gene expression changes for both cell lines. Eight pathways were identified to be recruited in the virus production state: amino acid catabolism, carbohydrate catabolism and integration of the energy metabolism, nucleotide metabolism, glutathione metabolism, pentose phosphate pathway, polyamines biosynthesis and lipid metabolism. Their ability to modulate viral titers was experimentally challenged, leading to improved specific productivities of recombinant retrovirus up to 6-fold. Within recruited pathways in the virus production state, we sought for metabolic engineering gene targets in the low producing phenotypes. A mining strategy was used alternative to the traditional approach 'high vs. low producer' clonal comparison. Instead, 'high vs. low producer' from different genetic backgrounds (i.e. cell origins) were compared. Several genes were identified as limiting in the low-production phenotype, including two enzymes from cholesterol biosynthesis, two enzymes from glutathione biosynthesis and the regulatory machinery of polyamines biosynthesis. This is thus a frontier work, bridging fundamentals to technological research and contributing to enlarge our understanding of enveloped virus production dynamics in mammalian cell hosts.

Scalable expansion of multipotent adult progenitor cells as three-dimensional cell aggregates.

Many applications of stem cell technologies require a large quantity of cells for which scalable processes of cell expansion and differentiation are essential. Multipotent adult progenitor cells (MAPCs) are adult stem cells isolated from the bone marrow with extensive self-renewal and broad differentiation capabilities. MAPCs are typically cultured surface adherent (2D) and at low cell density, making the large surface required for cell expansion a hindrance for many applications. This study demonstrates that MAPCs can be cultivated as aggregates in an undifferentiated state for at least 16 days, as levels of a number of transcripts, including Oct4, remained similar, Oct4 protein was unchanged, and differentiation to neural progenitor, endothelial cell and hepatocyte like cells was retained. Cultivation of these aggregates in stirred bioreactor lead to a 70-fold expansion in 6 days with final cell densities of close to 106/mL. Importantly, the MAPC aggregates recovered from stirred bioreactors could be differentiated to hepatocyte-like cells that expressed albumin, alpha-1-antitrypsin (AAT), and tyrosine amino transferase (TAT) transcripts and also secreted albumin and urea. This method of scalable expansion combined with differentiation of MAPCs can potentially be used for generating large numbers of MAPC and MAPC-derived differentiated cells.

Effect of air pollution on gout development: a nationwide population-based observational study.

OBJECTIVE: To investigate the effect of air pollution on gout development. METHODS: A total of 170318 participants were enrolled. These pollutants were considered: carbon monoxide (CO), fine particulate matter 2.5 (PM2.5), total hydrocarbons (THC) and methane (CH4). The yearly average concentrations were calculated from 2000 to 2011. Univariate and multivariate analyses by Cox proportional hazard regression models were adopted to estimate hazard ratios for gout in the Q2-Q4 concentrations of air pollutants compared with the Q1 concentration. RESULTS: In THC, relative to the Q1 concentration, the risk of gout was higher in participants exposed to the Q2-Q4 concentrations [adjusted hazard ratio (aHR), 1.10 with 95% confidence interval (CI), 1.01-1.19 in the Q2 concentration of THC; aHR, 4.20 with 95% CI, 3.93-4.49 in the Q3 concentration of THC; aHR, 5.65 with 95% CI, 5.29-6.04 in the Q4 concentration of THC]. In regard to CH4, when the Q1 concentration was defined as the reference, the risks of gout were increased for participants exposed to the Q2, Q3 and Q4 concentrations (aHR, 1.16 with 95% CI, 1.06-1.26 in the Q2 concentration of CH4; aHR, 2.37 with 95% CI, 2.20-2.55 in the Q3 concentration of CH4; aHR, 8.73 with 95% CI, 8.16-9.34 in the Q4 concentration of CH4). CONCLUSIONS: Association between air pollution and risk of gout was noted.

Effect of acupuncture on atrial fibrillation stratified by CHA2DS2-VASc score-a nationwide cohort investigation.

OBJECTIVE: This research aimed to make statements regarding the reduction in atrial fibrillation (AF) risk due to acupuncture, stratified by CHA2DS2-VASc score. METHODS: The Kaplan-Meier method was performed to calculate cumulative incidence of outcomes for each group, and the log-rank test were performed to compare differences between groups. Incidences and hazard ratios (HRs) were estimated by univariate Cox proportional hazards models, and adjusted HRs (aHRs) were estimated by multivariate Cox proportional hazards models including demographic covariates and comorbid status. RESULTS: In CHA2DS2-VASc scores of 0-1, 2-3, 4-5 and >5, cases with acupuncture were all associated with decreased incidence of AF (aHR 0.46 with 95% CI 0.42-0.51, P < 0.001 in the CHA2DS2-VASc scores of 0-1; aHR 0.53 with 95% CI 0.50-0.57, P < 0.001 in the CHA2DS2-VASc scores of 2-3; aHR 0.56 with 95% CI 0.52-0.61, P < 0.001 in the CHA2DS2-VASc scores of 4-5; and aHR 0.64 with 95% CI 0.55-0.74, P < 0.001 in the CHA2DS2-VASc scores of >5). CONCLUSION: Protective effect of acupuncture on AF was observed in this study, and the effect was more obvious for those with fewer comorbidities.

[Epidemic characteristics and dynamic changes of spatio-temporal distribution of hemorrhagic fever with renal syndrome in Guangzhou, 2010-2019].

Objective: To analyze the epidemic characteristics and spatio-temporal distribution of hemorrhagic fever with renal syndrome (HFRS) in Guangzhou from 2010 to 2019 and provide a basis for prevention and control strategies. Methods: The data of HFRS was from National Disease Reporting Information System and the epidemic investigation. A descriptive analysis was used. OpenGeoDa 1.2.0 software was used for global spatial autocorrelation and local spatial autocorrelation analysis. SatScan 9.6 software was used for detecting the hot spot area in time and space. ArcGIS 10.2 software was used for map visualization. Results: 1 298 cases of HFRS were reported, and three patients died in Guangzhou in 2010-2019. The annual incidence rate was 0.99/100 000. The proportion of 21-50 years old cases accounted for 70.88% and the male to female ratio was 2.98∶1. Most patients were house workers or unemployed, accounting for 31.28%, followed by business servants (accounting for 17.33%). The incidence peak in spring and winter accounted for 33.74% and 26.35% of the year. All districts reported cases in recent ten years. A total of 407 cases had been reported in Haizhu district, accounting for 31.36% of the total number of cases in the whole city. The annual incidence rate was 2.52/100 000. The number of reported cases and the annual incidence rate were the highest in Guangzhou. The clustered area showed that there was spatio-temporal clustering in Guangzhou. The aggregation area was mainly concentrated in the urban villages adjacent to Wan-mu orchard and the Haizhu Lake Wetland Park in Haizhu district (logarithmic likelihood ratio was 44.08, P<0.001). Conclusions: The prevalence and concentration of HFRS in winter and spring Guangzhou city from 2010 to 2019, showed a high incidence. Young and middle-aged men engaged in domestic and unemployed, and commercial services appeared the main risk groups. The urban-rural junction with many immigrants and low health environment, streets adjacent to Wan-mu orchard, and the Haizhu Lake Wetland Park in Haizhu district were the important regions for preventing and controlling HFRS. The government should formulate prevention and control measures to curb the rise and spread of the HFRS epidemic.

Retroviral recombination and reverse transcription.

Recombination occurs at a high rate in retroviral replication, and its observation requires a virion containing two different RNA molecules (heterodimeric particles). Analysis of retroviral recombinants formed after a single round of replication revealed that (i) the nonselected markers changed more frequently than expected from the rate of recombination of selected markers; (ii) the transfer of the initially synthesized minus strand strong stop DNA was either intramolecular or intermolecular; (iii) the transfer of the first synthesized plus strand strong stop DNA was always intramolecular; and (iv) there was a strong correlation between the type of transfer of the minus strand strong stop DNA and the number of template switches observed. These data suggest that retroviral recombination is ordered and occurs during the synthesis of both minus and plus strand DNA.

Micropatterning gradients and controlling surface densities of photoactivatable biomolecules on self-assembled monolayers of oligo(ethylene glycol) alkanethiolates.

BACKGROUND: Bioactive molecules that are covalently immobilized in patterns on surfaces have previously been used to control or study cell behavior such as adhesion, spreading, movement or differentiation. Photoimmobilization techniques can be used, however, to control not only the spatial pattern of molecular immobilization, termed the micropattern, but also the surface density of the molecules--a characteristic that has not been previously exploited. RESULTS: Oligopeptides containing the bioactive Arg-Gly-Asp cell-adhesion sequence were immobilized upon self-assembled monolayers of an oligo(ethylene glycol) alkanethiolate in patterns that were visualized and quantified by autoradiography. The amount and pattern of immobilized peptide were controlled by manipulating the exposure of the sample to a UV lamp or a laser beam. Patterns of peptides, including a density gradient, were used to control the location and number of adherent cells and also the cell shape. CONCLUSIONS: A photoimmobilization technique for decorating surfaces with micropatterns that consist of variable densities of bioactive molecules is described. The efficacy of the patterns for controlling cell adhesion and shape has been demonstrated. This technique is useful for the study of cell behavior on micropatterns.

Large-scale mammalian cell culture.

Mammalian cell culture continues to draw major research efforts. A great deal of progress has recently been made in cellular physiology, especially in factors adversely affecting cell growth or viability. Through molecular genetic manipulation, cells are more readily cultivated in a medium free of animal proteins. Achieving a high cell concentration and high viability continue to be common themes in engineering research. The need to implement a control policy for fed-batch and perfusion cultures has prompted increased efforts in process monitoring and control. Integrating these advances will be beneficial for ensuring product quality and process consistency.

Mammalian cell culture processes.

Over the past year, mammalian cell culture research has been aimed at investigating the influence of culture conditions on viability, productivity and the consistency of post-translational modifications. Studies of the effect of medium conditions and the development of kinetic models are being made in relation to current efforts to develop fed-batch strategies that will optimize recombinant protein production processes. Recent advances have included novel biosensor and bioreactor developments. New technologies have also been applied to investigate high cell density bioreactor and culture conditions.

Identification of an insertion-sequence-like genetic element in the newly recognized human pathogen Mycoplasma incognitus.

Cloned 2.2-kb DNA (plasmid psb-2.2) of Mycoplasma incognitus, a pathogen in AIDS and non-AIDS patients [Lo et al., Am. J. Trop. Med. Hyg. 41 (1989) 364-376; 601-616], contains a 1405-bp genetic element closely resembling bacterial insertion sequence (IS) elements. This IS-like element has 29-bp terminal inverted repeats with seven mismatches, is immediately flanked by 3-bp direct repeats, and has typical stem-and-loop structures at or near both the termini. Two potential open reading frames (ORF-1 and ORF-2) encode 143 amino acids (aa) and 103 aa, respectively, in this IS-like element. Part (57 aa) of the deduced aa sequence of ORF-2 has a significant homology (43%) with the putative transposase of Escherichia coli IS3. In this study, a series of synthetic oligodeoxyribonucleotides each containing a specific sequence of a selected segment in psb-2.2, have been used as probes which reveal that the IS-like element occurs more than ten times in the genome of M. incognitus. This potentially transposable element has many characteristic features in common with bacterial IS elements.

Identification of a putative infC-rpmI-rplT operon flanked by long inverted repeats in Mycoplasma fermentans (incognitus strain).

A specific 1542-bp DNA fragment was amplified from Mycoplasma fermentans (incognitus strain) using a unique 23-nucleotide (nt) synthetic deoxyribonucleotide (oligo) (5'-TCCAAAAAGTCCGGAATTTGGGG) as the primer pair in the polymerase chain reaction (PCR). The 23-nt sequence is part of the 29-bp terminal inverted repeat (IR) which forms the left potential stem-and-loop (s&l) structure of the previously identified M. fermentans insertion-sequence(IS)-like genetic element [Hu et al., Gene 93 (1990) 67-72]. The amplified DNA was cloned and sequenced. A pair of 27-bp IR containing the 23-nt synthetic oligo was identified at both termini. Between the IR, there are four potential open reading frames (ORFs) which are arranged adjacent to each other in the order, ORF-1, ORF-2, ORF-3 and ORF-4, with parts of ORF-1 and ORF-2 overlapping. The deduced amino acid (aa) sequences of ORF-2, ORF-3 and ORF-4 are 34 to 60% identical to the translation initiation factor IF3 (encoded by the infC gene), ribosomal proteins L35 (rpmI gene) and L20 (rplT gene) of Escherichia coli and Bacillus stearothermophilus, respectively. In bacteria, the infC-rpmI-rplT genes are organized to function as an operon. There are multiple sites with promoter-like sequences identified upstream from the putative infC gene in the mycoplasma closely resembling the gene arrangement in the bacterial operon. All three genes of ORF-2, ORF-3 and ORF-4 are preceded individually by a strong appropriately spaced (7 and 10 bp) putative Shine-Dalgarno sequence (5'-AAGGA).(ABSTRACT TRUNCATED AT 250 WORDS)

A technique for porcine hepatocyte harvest and description of differentiated metabolic functions in static culture.

Current bioartificial liver devices are based on the use of a large mass of hepatocytes exhibiting differentiated metabolic function. The pig has become a source of interest for the acquisition of such cells-however, harvesting a large mass of highly viable cells has met with difficulty. This study describes a technique for harvesting large quantities of hepatocytes at viabilities greater than 90% and also describes several features documenting differentiated function. Pigs, 6 to 10 kg body weight, underwent in situ two-step whole liver perfusion (ethylene glycol tetraacetic acid and collagenase) and ex vivo cell harvest. Harvests yielded an average of 19.5 billion cells with an average viability of 94.6%. Hepatocytes were then entrapped in type I collagen (3 x 10(5) cells/well) and cultured in serum-free media for 5 days. Pig hepatocytes produced stable amounts of albumin and maintained cytochrome P-450 and glucuronidation activity over 5 days, as shown by the metabolism of lidocaine and 4-methylumbelliferone. These data indicate that pig hepatocytes can be harvested with high yields and can retain viability and differentiated function over at least 5 days of culture, and therefore should prove to be an excellent source of hepatocytes for bioartificial liver devices.

Entrapment of hepatocyte spheroids in a hollow fiber bioreactor as a potential bioartificial liver.

A bioartificial liver (BAL) employing xenogeneic hepatocytes has been developed as a potential interim support for patients in hepatic failure. For application in human therapy, the BAL requires a substantial increase in liver-specific functions. Cultivation of hepatocytes as spheroids leads to enhanced liver specific functions. We explored the possibility of entrapping spheroids into the BAL in order to improve device performance. Rat hepatocyte spheroids were entrapped in collagen gel within the lumen fibers of the BAL. The morphology and ultrastructure of collagen-entrapped spheroids resembled those of suspended spheroids formed on petri dishes. Albumin synthesis and P-450 enzyme activity were measured as markers of liver specific functions of spheroids entrapped in the BAL. At least a 4-fold improvement in these functions was observed compared to BAL devices entrapped with dispersed hepatocytes in collagen gels.

Receding cytochrome P450 activity in disassembling hepatocyte spheroids.

Primary rat hepatocytes can self-assemble to form multicellular spheroids when plated onto Primaria petri dishes or suspended in stirred vessels. These spheroids exhibit prolonged viability, enhanced liver-specific functions and differentiated ultrastructure compared to monolayer cultures. Upon transfer to collagen coated surface, or upon the addition of fetal bovine serum (FBS) to the culture, these spheroids began to disassemble and spread on the surface. The dynamics of cytochrome P450 CYP1A1/2 activity in the course of spheroid disassembly was examined in situ by detection of the fluorescent product, resorufin, of ethoxyresorufin O-dealkylation. Optical sectioning of the disassembling spheroids by confocal microscopy demonstrated that hepatocytes that reverted to monolayer exhibited markedly lower CYP1A1/2 activity than those that remained in a multilayered structure. This occurred whether the disassembly was caused by incubation with FBS-containing medium or by cultivation on a collagen-coated surface. When spheroids were cultured on the surface of agar, the disassembly process was retarded even in the presence of FBS. However, even in those intact spheroids, the exposure to FBS markedly decreased CYP1A1/2 activity. The decreased CYP1A1/2 activity was correlated to a diminished smooth endoplasmic reticulum as seen in the transmission electron micrograph. The results clearly demonstrate that the disassembly of hepatocyte spheroids led to decreased CYP1A1/2 activity. Furthermore, FBS contained a factor that caused CYP1A1/2 to decrease even in intact spheroids.

Metabolic engineering of cephalosporin biosynthesis in Streptomyces clavuligerus.

The biosynthesis of beta-lactams is one of the most thoroughly studied antibiotic pathways. The availability of the characteristics and the time profiles of activities of enzymes involved in the biosynthesis allows one to critically evaluate the potential rate-limiting steps in its production. Our approach to understanding the control of beta-lactam biosynthesis has been pursued using a two-stage strategy: (1) to predict the rate-limiting steps using a kinetic model and (2) to relax the rate-limiting steps by engineering the biosynthetic pathway or by altering the kinetic parameters of the predicted key rate-limiting enzyme. Kinetic analysis of the pathway dynamics of cephamycin C production in Streptomyces clavuligerus was performed using data obtained from wild type. Sensitivity analysis revealed that the availability of precursor alpha-aminoadipic acid and activity of ACV synthetase were the potential rate-limiting steps. Relaxation of the precursor limitation was accomplished by integration of an additional copy of the gene encoding lysine-epsilon-aminotransferase (lat) into the chromosome. The recombinant strain showed an increased level of cephamycin C production as expected. The intracellular levels of different intermediates in the pathway in batch cultures were analyzed.

Enhanced cytochrome P450 IA1 activity of self-assembled rat hepatocyte spheroids.

Primary rat hepatocytes can self-assemble to form multicellular spheroids when plated onto Primaria petri dishes. Spheroids have been observed to exhibit enhanced liver-specific functions and differentiated ultrastructure compared to monolayer cultures on dry collagen. With confocal scanning laser microscopy, CYP1A1 activity was evaluated in situ by detecting resorufin. This highly fluorescent molecule is the P450-mediated product of 7-ethoxyresorufin O-dealkylation (EROD). Significantly higher P450 activity was observed in spheroids compared to monolayers on collagen upon induction with 50 microM beta-naphthoflavone (BNF), a CYP1A inducer. This was confirmed by measuring microsomal EROD activity. The distribution of CYP1A1 activity within spheroids was heterogeneous, with higher activity localized to the hepatocytes in the interior. During the process of spheroid formation, cells were initially seen to attach and spread out as a monolayer. This stage was associated with relatively low CYP1A1 activity. As cells formed multicellular structures and aggregated into spheroids, the level of CYP1A1 activity increased over time. At least a fivefold higher fluorescence intensity was observed in spheroids compared to that of monolayers maintained on collagen. The higher P450 activity within spheroids may be associated with their ability to maintain a greater degree of differentiation compared to monolayers. These studies demonstrate the potential of hepatocyte spheroids as a model system for investigating drug metabolism, tissue engineering, and tissue self-assembly.