INHIBITION OF MYOBLAST FUSION AFTER ONE ROUND OF DNA SYNTHESIS IN 5-BROMODEOXYURIDINE

The thymidine analogue 5-bromodeoxyuridine (BUdR) has a differential effect on the synthesis of tissue-specific products and molecules required for growth and division. Proliferating myogenic cells cultured in BUdR fail to fuse and fail to initiate the synthesis of contractile protein filaments. Conversely, BUdR has but a minor effect on cell viability and reproductive integrity. Low concentrations of BUdR result in an enhancement of cell number relative to the controls; higher concentrations are cytotoxic. Suppression of myogenesis is reversible after at least 10 cell generations of growth in the analogue. Cells that do not synthesize DNA, such as postmitotic myoblasts and myotubes, are not affected by BUdR. Incorporation of BUdR for one round of DNA synthesis was accomplished by first incubating myogenic cells, prior to fusion, in 5-fluorodeoxyuridine (FUdR) to block DNA synthesis and collect cells in the presynthetic phase. The cells were then allowed to synthesize either normal DNA or BU-DNA for one S period by circumventing the FUdR block with BUdR or BUdR plus thymidine (TdR). The cultures were continued in FUdR to prevent dilution of the incorporated analogue by further division. After 3 days, the cultures from the FUdR-BUdR series showed the typical BUdR effect; the cells were excessively flattened and few multinucleated myotubes formed. Cells in the control cultures were of normal morphology, and multinucleated myotubes were present. These results were confirmed in another experiment in which BUdR-³H was added to 2-day cultures in which myotubes were forming. Fusion of thymidine-³H-labeled cells begins at 8 hr after the preceding S phase. In contrast, cells which incorporate BUdR-³H for one S period do not fuse with normal myotubes.     

Altered glycosaminoglycan production in cultured osteogenesis-imperfecta skin fibroblasts.

Collagen and glycosaminoglycan syntheses were studied in skin fibroblasts cultured from patients with osteogenesis imperfecta (OI) and from age-matched controls. Collagen synthesis (measured as protein-bound [3H]hydroxyproline) was decreased in all four OI cell lines studied in the present experiments, comprising 16-24% of total protein synthesis (40% in normal cells). Hyaluronic acid production in OI skin fibroblasts per cell was higher than in age-matched controls, but the production of sulphated glycosaminoglycans was at the normal level. Thus the ratio of the hyaluronic acid and sulphated-glycosaminoglycan radioactivities was markedly higher in OI cultures than in control cultures, especially at the exponential phase of growth where the synthesis of hyaluronic acid was highest. Hyaluronic acid in OI had a normal molecular weight when determined by gel filtration on Sepharose 2B. The removal of high-molecular-weight hyaluronic acid from the medium by hyaluronidase had no effect on the rate of collagen secretion in OI cell line 1 (A.T.C.C. 1262), in which the rate of collagen secretion was lowest.     

COMPARISON OF THE GROWTH AND DIFFERENTIATION OF EPITHELIAL CELLS FROM NORMAL AND HYPERPLASTIC LENSES OF THE CHICK: STUDIES OF IN VITRO CELL CULTURES

Epithelial cells from hyperplastic lenses of a strain of chicks (Hy-1) selected for high growth rate were dissociated and cultured in vitro and compared with lens epithelial cells from a normal strain (N) in similar conditions. The hyperplastic lens cells showed remarkable motility and adhesiveness after dissociation and formed cell aggregates of various sizes before attaching to the substrate, giving a rather low plating efficiency. The lens structures (lentoid bodies) developed in partially confluent cultures of Hy-1 cells at least three days earlier than those in the cultures from normal control cells, in which the lens structures developed only after the cultures reached confluence. The results of culture at low cell density showed that the Hy-1 cell population consisted of at least two cell types different from each other in growth capacity. These striking differences in in vitro behaviour of dissociated cells from normal and hyperplastic lens epithelia and the results of clonal culture are discussed in relation to the possible mechanisms of abnormal morphogenesis and growth which are likely to be involved in the development of the hyperplastic lens in situ .     

Enhancement of photosynthetic O2 evolution in Chlorella vulgaris under high light and increased CO2 concentration as a sign of acclimation to phosphate deficiency.

The photosynthetic oxygen evolution of Chlorella vulgaris (Beijer.) cells taken from phosphate-deficient (-P) and control cultures was measured during 8 days of culture growth. Under inorganic carbon concentration (50 microM) in the measuring cell suspension and irradiance (150 micromol m(-2) s(-1)), the same as during culture growth, there were no marked differences in the photosynthetic O2 evolution rate between the -P cells and the controls. The much slower growth of -P cultures indicated that the utilization of absorbed photosynthetically active radiation (PAR) in the CO2 assimilation and biomass production were in -P cells less efficient than in the controls. Alga cells under the phosphorus stress utilized more of the absorbed PAR in the nitrate reduction than the control cells. However, under conditions of more efficient CO2 supply (inorganic carbon concentration 150 microM, introducing of exogenous carbonic anhydrase to the measuring cell suspension) and under increased irradiance (500 micromol m(-2) s(-1)), the photosynthetic O2 evolution in -P cells reached a higher rate than in the controls. The results suggest that in -P cells the restricted CO2 availability limits the total photosynthetic process. But under conditions more favorable for the CO2 uptake and under high irradiance, the -P cells may reveal a higher photosynthetic oxygen evolution rate than the controls. It is concluded that an increased potential activity of the photosynthetic light energy absorption and conversion in the C. vulgaris cells from -P cultures is a sign of acclimation to phosphorus stress by a sun-type like adaptation response of the photosynthetic apparatus.     

Induction of reverse transformation and normal cell cycle regulation by dibutyryl cAMP in a chemically transformed cell line

The objective of this study was to determine whether N6, O2-dibutyryl 3',5'-adenosine monophosphate (db-cAMP)-induced reverse transformation in a chemically transformed mouse cell line, AKR-MCA, would restore normal cell cycle regulation, particularly with regard to their growth arrest in the early G1 period. The AKR-MCA cells were grown to confluency in the presence or absence of db-cAMP (0.5 mM) plus theophylline (1 mM). The confluent cultures were trypsinized and a portion of the cells were fused with mitotic HeLa cells to induce premature chromosome condensation, while the remaining cells were used to study the kinetics of initiation of DNA synthesis. The prematurely condensed chromosomes (PCC) of the control and the treated cultures were classified into G1, S, or G2 types on the basis of their morphology. The G1 PCC were further subclassified into six groups (+1 - +6); +1 being the most condensed and +6 the most decondensed. The cyclic AMP (cAMP)-treated cells exhibited better attachment to the culture dish, were blocked in early G1 period at confluency, and entered S phase about 4 h later than the control following subculturing. In contrast, a majority of cells in the control cultures were arrested in S phase at confluency. These data indicate that the db-cAMP-induced reverse transformation in AKR-MCA cells at least partially restores normal cell cycle regulation in these chemically transformed cells.     

Effects of retinoic acid on cell proliferation and cell differentiation in a rat tracheal epithelial cell line

Abstract. The effects of 3.3 times 10-⁷ M to 3.3 times 10^-5 M all- trans -retinoic acid (vitamin-A acid) on the total cell population dynamics of 165 S, a keratinizing epithelial cell line from carcinogen-exposed rat trachea, were studied. During the first 6 days of culture, cells accumulated on the dish in the presence of the vitamin to twice the density of controls. [³H]thymidine incorporation into DNA and percentage of [³H]thymidine-labelled cells in autoradiographs were stimulated in a dose-dependent fashion to a maximum of 25- and 34-fold, respectively. Exfoliation of cells from the cultures was also enhanced 2–3-fold, resulting in nearly twice the total number of cells (attached plus exfoliated) in the presence of the vitamin.
During 19 days of culture, retinoic acid maintained a higher level of [³H]thymidine incorporation and cell exfoliation in 165 S cells so that by day 19, total cell production was more than three times that seen in controls. At this time, vitamin-treated cultures showed a reduced cell saturation density compared to controls. The higher final cell density in the control cultures was a result of multilayering and papillary formation which did not occur in the presence of retinoic acid. The papillae in control cultures stained specifically with Rhodamine B or with the eosin and orange G components of the Papanicolaou method. A count of the number of eosin and orange G positive cells in the attached and exfoliated cell compartments showed an 8-fold reduction of keratinization in retinoic acid-exposed cultures. Our results show that retinoic acid is a growth stimulant in these cell cultures, causing increased cell proliferation and exfoliation accompanied by inhibition of keratinization.     

Proliferative kinetics and mitomycin C-induced chromosome damage in Fanconi's anemia lymphocytes

Lymphocytes from two sisters with Fanconi's anemia (FA) were studied for cell cycle kinetics, sister chromatid exchanges (SCEs), and chromosomal aberrations when they had undergone one, two, or three or more divisions in mitomycin C (MMC)-treated cultures. Lymphocytes from the parents, another sister of the probands, and a healthy unrelated adult were examined as controls. Analyses of cell cycle kinetics by the sister chromatid differential staining method revealed that the relative frequency of metaphase cells at their third or subsequent divisions was much smaller in untreated FA cultures than in normal cultures fixed at 96 h after phytohemagglutinin stimulation. These data indicate that FA cells proliferate much more slowly than normal cells. MMC treatments of FA and normal cells led to a clearly dose-related delay in cell turnover times, the duration of delay being much longer in FA than in normal cells. FA cells had about 1.4 times higher frequencies of SCEs than normal cells in both MMC-treated and untreated cultures. FA cells also showed several times higher frequencies of chromosomal aberrations than normal cells, and the frequency of chromosomal aberrations decreased through subsequent mitoses by approximately 60% in both FA and normal cells.     

Regulation of synthesis of a brain-specific protein in monolayer cultures of clonal rat glial cells

C6 cells were grown in monolayer culture under conditions permitting continued exponential cell division after attainment of a density at which extensive intercellular contacts were formed. An increase in the relative synthesis of S100 protein coincided with the time of formation of extensive intercellular contacts and preceded the onset of the stationary phase of growth by three generations. These observations suggested that the induction of S100 protein synthesis was mediated by cell contact and not by an arrest of cellular growth. The mechanism of this induction was first studied in a homologous non-initiating cell-free protein-synthesizing system from C6 cells, using fixed amounts of free amino acids or fully charged rat liver aminoacyl-tRNA as a source of precursors for protein synthesis. Real synthesis of total soluble proteins decreased as the cells progressed from logarithmic to stationary growth while synthesis of S100 protein increased during this period. The capacity of poly(A)+ RNA from logarithmic and stationary cultures to direct the synthesis of S100 protein was estimated in a cell-free protein-synthesizing system derived from wheat embryos. Increased synthesis of S100 protein in stationary cultures was directly correlated with an increase in translatable S100 protein mRNA.     

Control of the uptake of amino acids by serum in chick embryo cells,untransformed or transformed with rous sarcoma virus

Summary Forty to fifty minutes after removal of serum, the net total uptake of amino acids in growing secondary cultures of normal or virus-transformed chick embryo cells, stopped or proceeded only at a highly reduced rate. In both normal and transformed cells, theinitial (0–40 min) rate of the above uptake was the same in the absence of serum as in its presence. The initial rate of the total uptake of amino acids in growing transformed cells was about the same as in growing normal cells. Neither in the normal nor in the transformed cells was the rate of the total uptake of amino acids reduced by cell confluence alone. In highly dense, hyperconfluent cultures of normal cells in which cell growth was arrested, the rate of uptake in the absence or in the presence of serum was four- to fivefold lower than the rate obtained in growing normal cells under similar conditions; in the absence of serum, the net uptake stopped after 40 min in the hyperconfluent cultures as well. It appears that cells growing in tissue culture require a serum factor for maintenance of the required high rates of uptake of amino acids and that the inhibition of growth at high cell densities is a result of depletion of this factor from serum, or the inability of the cells in a dense culture to respond to the factor. A serum factor is apparently also required for maintenance of the reduced rates of uptake of amino acids observed in hyperconfluent cultures.     

Cell cycle parameters of slowly growing Escherichia coli B/r studied by flow cytometry

The cell cycle kinetics of Escherichia coli B/r A and B/r K cells were studied by flow cytometry. Three-dimensional histograms of cell cultures show the number of cells as a function of cellular DNA and protein contents and give detailed pictures of the cell cycle distribution with regard to these parameters. Histograms of slowly growing chemostat cultures showed that cell cycle periods B and C + D increase with a decreasing growth rate and that the B period occupies an increasing fraction of the cycle. The DNA replication patterns of B/r A and K were found to be quite similar. At extremely low growth rates (doubling time [T] = 17 h), B/r A cells had a B period of 0.8 T, a C period of 0.1 T, and a D period of 0.1 T, and B/r K cells (T = 16 h) had a B period of 0.6 T, a C period of 0.15 T, and a D period of 0.25 T. Mass increase, i.e., essentially protein synthesis, was seen in all three periods of the cell cycle. For B/r A cells, the average rate of mass increase was 11 times greater in the D period than in the B period, whereas for B/r K cells the rate of mass increase was twice as great in the D period as in the B period. The DNA and cell size distributions of batch cultures in exponential growth were found to vary with time, indicating that such cultures are not suitable for studies of cell cycle kinetics.     

Effect of incubation temperature on the frequency of sister chromatid exchanges in Bloom's syndrome lymphocytes

Summary The effect of incubation temperature on the frequency of sister chromatid exchange (SCE) has been studied in blood cultures from three Bloom's syndrome (BS) patients, three controls, and three BS heterozygotes. All cell types show slight increases of SCE at 39°C while at 35°C and 32°C, SCE is reduced considerably in BS and slightly increased in normal cells. Prolonging lymphocyte culture to 140 h and adding BUdR for the last two S periods causes a similar decrease in the percentage of SCE in normal and BS cells but, while the latter show a further reduction if they are incubated at 32°C during BUdR labelling, the normal cells show an increase. Therefore, BS and control lymphocytes respond similarly to changes in incubation time and differently to changes in incubation temperature. The possibility that the discrepant behaviour of the BS and control cultures may be due to different growth kinetics of their B and T lymphocytes has been discussed but considered unlikely. Since low temperature lengthens the cell cycle, it has been suggested that our findings and those published by others on co-cultivation experiments (except those of Tice et al. 1978) can be explained by assuming that slow growth reduces SCE in BS cells. This, and unpublished observations (Giannelli et al. 1981), suggest that some imbalance in the factors responsible for DNA replication may exist in BS and possibly account for the high level of SCE.     

Effect of interferon on growth and division cycle of Friend erythroleukemic murine cells in vitro

The administration of appropriate doses of interferon to cultures of Friend leukemia cells causes a pronounced inhibition of cell growth. Several lines of evidence indicate that this effect is due to interferon itself, rather than to unknown contaminants of interferon preparations. Autoradiograph analysis of growth parameters of Friend leukemia cells during treatment with interferon demonstrates that the rate of entry into the S phase, the percent decline of unlabeled mitoses, and the mitotic indexes are significantly lower in interferon- treated cell cultures than in control untreated cultures when tritiated thymidine was added 12 h after the administration of interferon. These data indicate that fractions of interferon-treated cell population are delayed in both G1 and in G2 phases of the cell cycle. This was confirmed by exact measurements of the length of the various phases of the cycle. The interferon-induced inhibition of growth of Friend leukemia cells is reversible after removal of the compound. Autoradiograph data obtained from control cultures and from cultures previously treated with interferon that had been washed free of interferon and reseeded in interferon-free medium, demonstrate that during the first 12 h after removal of interferon, a large majority of the cells previously treated with interferon had a deranged flow into the S phase, a high number of unlabeled mitoses, and a low mitotic index. These data provide further evidence for the above-mentioned prolongations of G1 and G2 phases of the cell cycle. All growth parameters tested reverted to normal values within 12 h after washing out interferon.     

Outgrowth of fibroblast cells from goat skin explants in three different culture media and the establishment of cell lines

Three different commercially available media, known to support human and porcine-specific fibroblast cultures, were tested for their growth potential on goat skin explants. Although outgrowth of fibroblasts was observed in all media tested, irrespective of breed, porcine-specific media exhibited higher rate of growth. Using this media, three fibroblast cell lines (GSF289, GSF737, and GSF2010) from ear skin explants of normal healthy dairy goats of Kiko and Saanen breed were successfully established in culture. Liquid nitrogen stocks of these frozen cells had a viability rate of 96.2% in in vitro cultures. These cells were morphologically indistinguishable from the cell stocks prior to freezing. Analysis of the growth of a fifth passage culture revealed an ‘S’ shaped growth curve with a population doubling time of 25 h. The cell lines were found negative for microbial, fungal, and mycoplasma contaminations. These goat skin fibroblast lines and the simple method of their isolation and freezing with high rate of viability will provide additional tools to study molecular mechanisms that regulate fibroblast function and for genetic manipulation of small ruminants.     

Hyaluronan structures synthesized by rat mesangial cells in response to hyperglycemia induce monocyte adhesion

Mesangial expansion, the principal glomerular lesion in diabetic nephropathy, is preceded by a phenotypic activation and transient proliferation of the glomerular mesangial cells and by a prominent glomerular infiltration of monocytes and macrophages. Because this infiltration seems to play a key role in the subsequent mesangial matrix expansion, we tested the response of cultures of rat mesangial cells (RMCs) for monocyte adhesion in response to hyperglycemia. Increasing the medium glucose concentration from 5.6 mm (normal) to 25.6 mm (hyperglycemic) significantly increased hyaluronan in the cell matrix, with a concurrent 3- to 4-fold increase in adhesion of U937 monocytic leukemic cells to cultures of near confluent RMCs. These responses were attributed directly to the high glucose concentration and not to increased extracellular osmolality. The monocytes primarily bind directly to hyaluronan-based structures in vitro. Abnormal deposits of hyaluronan were found in glomeruli of kidney sections from diabetic rats 1 week after streptozotocin treatment, often with closely associated monocytes/macrophages, suggesting that similar structures are relevant in vivo. The monocyte adhesion response to high glucose concentration required growth stimulation of RMCs by serum and activation of protein kinase C, and was inhibited by prior passage of the RMCs in the presence of heparin. These results suggest that the response may be cell growth state and protein kinase C-dependent. When incubated with the viral mimetic, poly I:C, in the presence of normal glucose, heparin-passaged RMCs still increased cell-associated hyaluronan and exhibited hyaluronan-mediated adhesion of monocytes, indicating that the two stimuli, high glucose and viral mimetic, induce the production of the hyaluronan structures that promote monocyte adhesion by distinctly different intracellular signaling mechanisms.     

Growth stimulation of 3T3 fibroblasts by cystatin

Treatment of cultures of mouse 3T3 fibroblasts with Cystatin C, a thiol-proteinase inhibitor isolated from chicken egg white, resulted in an enhanced rate of cell proliferation. This stimulation was demonstrated using two independent assay systems: (a) assessment of total cell number and (b) measurement of [3H]thymidine incorporated into acid-precipitable DNA. In both assays, the dose-response curves of Cystatin stimulation showed a rising function that plateaued at a concentration of approximately 120 micrograms/ml. The addition of Cystatin to cultures of Kirsten murine sarcoma virus-transformed 3T3 cells also enhanced DNA synthesis in these target cells. Control experiments showed that the presence of Cystatin did not alter the level of binding of radioactively labeled epidermal growth factor and platelet derived growth factor to 3T3 cells. These results argue against the possibility that the observed growth stimulation by Cystatin was due to growth factor contamination of the Cystatin preparation.     

Growth Characteristics, Bile Sensitivity, and Freeze Damage in Colonial Variants of Lactobacillus acidophilus

Rough (R) and smooth (S) colonial variants were isolated from a heterogeneous culture of Lactobacillus acidophilus RL8K. R and S types were stable upon repeated transfer on agar, but revertant colonies did appear after broth transfers. When propagated in commercial MRS broth, R and S cultures showed similar growth characteristics, and both cell types were insensitive to freezing and frozen storage at −20°C. Alternatively, during growth in scratch MRS broth, R cultures shifted to a reduced rate of growth during the late logarithmic phase. R cells grown under these conditions were susceptible to death by freezing and injury at −20°C. Microscopically, R cells were observed as long gram-positive rods with small nonstainable blebs protruding from the cell wall. In bile sensitivity studies of R and S cells plated on MRS agar plus oxgall, the S culture was resistant to 1% bile, whereas the R culture was sensitive to 0.6% bile. Differences in the bile resistance and freeze damage of R and S cells suggest that colonial and cellular morphologies are important considerations for the selection of Lactobacillus strains as dietary adjuncts and for the development of growth conditions for preparing frozen concentrated cultures from either cell type.     

The role of calmodulin in the proliferation of transformed and phenotypically normal tsASV-infected rat cells

NRK cells infected with a temperature-sensitive, transformation-defective mutant of avian sarcoma virus (ASV), tsLA23, behaved as if nontransformed at a nonpermissive 40 degrees C and were rendered quiescent by serum deprivation. These serum-deprived cells were stimulated to start entering S phase about 7 hours after serum addition at 40 degrees C or about 9 hours after shifting the cultures to 36 degrees C, a temperature allowing the production of active viral pp60src and expression of the transformed phenotype. The transit of both serum- and temperature-stimulated tsLA23-NRK cells through later G1 was inhibited by the unrelated calmodulin antagonists W7 and R24571. The former drug was found to block the cells at a point in the cell cycle no more than 2 hours from the G1/S transition. The weaker calmodulin antagonist, W5, was less effective in impairing progression. Thus, calmodulin is likely required for the transit of both transformed and phenotypically normal tsLA23-NRK cells through the later stages of their G1 phases. Cells neoplastically transformed by ASV contain more calmodulin than uninfected, non-neoplastic cells. At the nonpermissive 40 degrees C, the calmodulin content of the tsLA23-NRK cells dropped to the non-neoplastic level. When these phenotypically nontransformed cells were enabled to reenter the cell cycle while still in low-serum medium by a 40 to 36 degrees C shift, they passed through the G1 and S phases and divided without a concomitant rise in the total calmodulin content. Thus, a calmodulin rise does not appear to be required for the expression of one characteristic of transformed cells, i.e., reduced requirement for exogenous growth factors.     

Relationship between cell growth and viral antigen content of cultures of Nicotiana tabacum inoculated with tobacco mosaic virus

Cell suspension cultures of Nicotiana tabacum (cv. White Burley)were inoculated with TMV (type strain) by coincubation. Infectionof the cells occurred over the 24 h period following inoculation.Over the interval 2–6 d following inoculation, which correspondedto the period of mitotic activity of the cultures, the viralantigen and infectivity of the cultures declined. The resultssuggest a link between host cell division and loss of virusfrom the cells. This decline was accompanied by the appearancein the culture extracts of virus rods of less than normal (300nm) length, and an overall reduction in the total number ofvirus particles per field under the electron microscope. Theseobservations suggested that the loss of infectivity of the extractsresulted, at least in part, from disruption of the virus. Afterthe end of the period of mitosis, the cultures showed a secondphase of antigen accumulation. However, the infectivity of theextracts continued to decrease to zero over the same periodand the proportion of shortened virus rods increased. Infectionby TMV altered the growth of the tobacco cells. Over the first6 d following inoculation, the growth rate was reduced, whereasfollowing day 6, the growth rate of inoculated cultures washigher than uninfected controls. Key words: Cell division, plant cell culture, plant virus, resistance, tobacco mosaic virus     

Infection of immune mast cells by Harvey sarcoma virus: immortalization without loss of requirement for interleukin-3.

Cells from adult mouse spleens were cultured in WEHI-3 cell-conditioned medium, which contains the lymphokine interleukin-3 (IL-3). Under these conditions, cells grow well for 4 to 8 weeks; the cultures contain a variety of cell types for the first 1 to 2 weeks but are subsequently composed largely of immune mast cells. We found that infection of these cultures with Harvey sarcoma virus (HaSV) profoundly enhanced the growth potential of the cells, resulting in the reproducible isolation of long-term cell lines. These HaSV-infected cells appeared to be phenotypically identical to the immune mast cells found in uninfected cultures as determined by biochemical, immunological, and cytological tests. Although the cells expressed protein p21Ha-ras at levels similar to those in HaSV-transformed fibroblasts, they continued to require IL-3 for growth in vitro. Similar IL-3-dependent, long-term mast cell lines were also cultured from the enlarged spleens present in HaSV-infected mice. These results suggest that high-level expression of an activated Ha-ras oncogene enhances growth in these cells, perhaps by stimulating the progression of the cells into S, without affecting differentiation or altering the requirements for normal growth factor.     

High-titer adenovirus vector production in 293S cell perfusion culture

Human 293S cells culture for recombinant adenovirus production is traditionally carried out in batch at a maximum of 6 x 10(5) cells/mL. A previous report demonstrated that fed-batch, applied to the adenovirus/293S cells system, improves the volumetric production of viral proteins by increasing the cell density at which cells can be infected, up to 2 x 10(6) cells/mL, without reducing the per-cell yield of product. To increase this cell density limit, the adenovirus production was performed in a perfusion system where the cells were separated by means of a tangential flow filtration device. 293S cell growth to 14 x 10(6) cells/mL was achieved in 10 days, at a medium renewal rate of 1 volume of medium per reactor volume and day (VVD). For adenovirus production, three 293S cell cultures were perfused at 1 VVD in parallel and infected at an average density of 8 x 10(6) cells/mL. One of the cultures was set at 37 degrees C and the two others at 35 degrees C. After a rapid initial cell loss, the average cell density stabilized at 5.75 x 10(6) cells/mL, 12 h postinfection, which was 8 times higher than the cell density in the batch control. This allowed the production of 3.2 x 10(9) infectious viral particles/mL (IVP/mL) at 37 degrees C and 7.8 x 10(9) IVP/mL at 35 degrees C, this last result being 5.5 times higher than the control. To our knowledge, this nonconcentrated titer is the highest value that has ever been published for adenovirus vector production. These observations lead to the conclusion that perfusion is an efficient tool to maintain, at high cell density, a specific production rate level sufficient to increase significantly the adenovirus volumetric production. Furthermore, it shows that perfusion at 35 degrees C can improve viral titer by 2.4-fold compared to 37 degrees C, in accordance with a previous study on adenovirus batch production.