High-pressure inactivation of dried microorganisms

Dried microorganisms are particularly resistant to high hydrostatic pressure effects. In this study, the survival of Saccharomyces cerevisiae was studied under pressure applied in different ways. Original processes and devices were purposely developed in our laboratory for long-term pressurization. Dried and wet yeast powders were submitted to high-pressure treatments (100-150 MPa for 24-144 h at 25 degrees C) through liquid media or inert gas. These powders were also pressurized after being vacuum-packed. In the case of wet yeasts, the pressurization procedure had little influence on the inactivation rate. In this case, inactivations were mainly due to hydrostatic pressure effects. Conversely, in the case of dried yeasts, inactivation was highly dependent on the treatment scheme. No mortality was observed when dried cells were pressurized in a non-aqueous liquid medium, but when nitrogen gas was used as the pressure-transmitting fluid, the inactivation rate was found to be between 1.5 and 2 log for the same pressure level and holding time. Several hypotheses were formulated to explain this phenomenon: the thermal effects induced by the pressure variations, the drying resulting from the gas pressure release and the sorption and desorption of the gas in cells. The highest inactivation rates were obtained with vacuum-packed dried yeasts. In this case, cell death occurred during the pressurization step and was induced by shear forces. Our results show that the mechanisms at the origin of cell death under pressure are strongly dependent on the nature of the pressure-transmitting medium and the hydration of microorganisms.     

Trypanosoma cruzi: morphogenesis in diffusion chambers in the mouse peritoneal cavity and attempted stimulation of host immunity

Sequential morphologic changes and antigen producing capacity of Trypanosoma cruzi in peritoneally implanted diffusion chambers were studied. Diffusion chambers were equipped with two Nuclepore filters (0.20 μm pore size) sandwiched between three Lucite rings. Epimastigotes or trypomastigotes and amastigotes were placed in diffusion chambers and surgically implanted into the peritoneal cavity of mice, or placed in in vitro cell culture, or in various types of culture media and incubated at 26 or 37 C.Epimastigotes maintained in diffusion chambers in mice changed into trypomastigotes as evidenced by the presence of numerous transitional stages and the concomitant decrease in the percentage of the former and increase in the percentage of the latter in chambers removed and examined at 16, 24, 36, 48, 72, and 84 hr after implantation. The maximum of 68% trypomastigotes was noted in chambers examined at 84 hr. Amastigotes subsequently appeared, apparently arising from trypomastigotes and reached the highest percentage (49%) obtained at 132 hr. The total number of parasites in chambers decreased slightly during the first 36 hr (20%). Little change in the total number of parasites was noted during the interval of 36–108 hr. A subsequent decrease in numbers of parasites was noted until by 280 hr after implantation, chambers contained less than 2% of the original number of organisms present in the chambers. No similar transformation of epimastigotes was noted in diffusion chambers maintained in cell culture at 37 C or in a cell culture growth medium or LIT medium at 37 or 26 C.No detectable morphological change was noted when trypomastigotes and amastigotes were implanted in diffusion chambers in the peritoneal cavity of mice. The total number of these parasites decreased notably (82%) after 24 hr.Mice receiving diffusion chambers containing epimastigotes implanted at two different intervals (21 days apart), developed only marginal protective immunity when challenged with virulent T. cruzi three weeks after the second implant of chambers, and no protection was afforded those mice implanted with chambers containing trypomastigotes and amastigotes. Sera collected from mice 6 wk after the second implantation of diffusion chambers containing parasites were observed to have antibody titers to T. cruzi as demonstrated by the fluorescent antibody technique and direct agglutination procedure.     

Characterization of Recombination Effects in a Liquid Ionization Chamber Used for the Dosimetry of a Radiosurgical Accelerator

Most modern radiation therapy devices allow the use of very small fields, either through beamlets in Intensity-Modulated Radiation Therapy (IMRT) or via stereotactic radiotherapy where positioning accuracy allows delivering very high doses per fraction in a small volume of the patient. Dosimetric measurements on medical accelerators are conventionally realized using air-filled ionization chambers. However, in small beams these are subject to nonnegligible perturbation effects. This study focuses on liquid ionization chambers, which offer advantages in terms of spatial resolution and low fluence perturbation. Ion recombination effects are investigated for the microLion detector (PTW) used with the Cyberknife system (Accuray). The method consists of performing a series of water tank measurements at different source-surface distances, and applying corrections to the liquid detector readings based on simultaneous gaseous detector measurements. This approach facilitates isolating the recombination effects arising from the high density of the liquid sensitive medium and obtaining correction factors to apply to the detector readings. The main difficulty resides in achieving a sufficient level of accuracy in the setup to be able to detect small changes in the chamber response.     

Air pressure in clamp-on leaf chambers: a neglected issue in gas exchange measurements

Air pressure in leaf chambers is thought to affect gas exchange measurements through changes in partial pressure of the air components. However, other effects may come into play when homobaric leaves are measured in which internal lateral gas flow may occur. When there was no pressure difference between the leaf chamber and ambient air (DeltaP=0), it was found in previous work that lateral CO(2) diffusion could affect measurements performed with clamp-on leaf chambers. On the other hand, overpressure (DeltaP>0) in leaf chambers has been reported to minimize artefacts possibly caused by leaks in chamber sealing. In the present work, net CO(2) exchange rates (NCER) were measured under different DeltaP values (0.0-3.0 kPa) on heterobaric and homobaric leaves. In heterobaric leaves which have internal barriers for lateral gas movement, changes in DeltaP had no significant effect on NCER. For homobaric leaves, effects of DeltaP>0 on measured NCER were significant, obviously due to lateral gas flux inside the leaf mesophyll. The magnitude of the effect was largely defined by stomatal conductance; when stomata were widely open, the impact of DeltaP on measured NCER was up to 7 mumol CO(2) m(-2) s(-1) kPa(-1). Since many other factors are also involved, neither DeltaP=0 nor DeltaP>0 was found to be the 'one-size fits all' solution to avoid erroneous effects of lateral gas transport on measurements with clamp-on leaf chambers.     

A combined approach for the enhanced detection and isolation of Bartonella species in dog blood samples: pre-enrichment liquid culture followed by PCR and subculture onto agar plates

Historically, direct plating, lysis centrifugation, or freeze-thaw approaches have proven to be highly insensitive methods for confirming Bartonella species infection in dogs. A prospective study was designed to compare diagnostic methods for the detection of Bartonella using samples submitted to the Vector-Borne Disease Diagnostic Laboratory at North Carolina State University. Methods included indirect immunofluorescence assay, PCR, direct inoculation of a blood agar plate (trypticase soy agar with 5% rabbit blood), and inoculation into a novel pre-enrichment liquid medium, Bartonella/alpha-Proteobacteria growth medium (BAPGM). Sequential research efforts resulted in the development of a combinational approach consisting of pre-enrichment culture of Bartonella species in BAPGM, sub-inoculation of the liquid culture onto agar plates, followed by DNA amplification using PCR. The multi-faceted approach resulted in substantial improvement in the microbiological detection and isolation of Bartonella when compared to direct inoculation of a blood agar plate. Importantly, this approach facilitated the detection and subsequent isolation of both single and co-infections with two Bartonella species in the blood of naturally infected dogs. The use of a combinational approach of pre-enrichment culture and PCR may assist in the diagnostic confirmation of bartonellosis in dogs and other animals.     

A spin label study of erythrocyte membranes during simulation of freezing

Summary Human erythrocytes were labeled with stearic acid spin labels, and no change was detected in membrane fluidity under hyperosmotic stress, going from isotonicity to about 3000 mOsm. Intact erythrocytes labeled with an androstane spin label and submitted to simulation of freezing show the onset of irreversible structural breakdown occurring in a saline solution at 2,000 mOsm. Ghosts labeled with maleimide spin label (4-maleimide-2,2,6,6-tetramethylpiperidinooxyl) when submitted to solutions of increasing osmolalities (pH 7.4), exhibit protein conformational changes that are irreversible after a simulated freeze-thaw cycle. After sonication of maleimide spin-labeled ghosts, membrane buried sulfhydryl groups become exposed. Such preparations showed behavior similar to the unsonicated when in saline hyperosmolal medium (pH 7.4). Such results suggest the ionic strength of the medium as the determining factor of the detected conformational changes. Maleimide spin-labeled ghosts in 300 mOsm saline solution (pH 7.4) were treated with ascorbic acid (spin destruction of nitroxides), and the kinetic analysis indicates that 65% of the labeled sites are located at the external interface of the membrane or in hydrophilic channels. Deformation and rearrangements of membrane components in solutions of increasing osmolalities apparently are related to protein conformational changes, on the outside surface of erythrocyte membranes, with a significant amount being structurally dissociated of lipids.     

Podospora anserina does not senesce when serially passaged in liquid culture.

A procedure was developed for the prolonged growth of the ascomycete fungus Podospora anserina in liquid culture to determine the effects of such growth on the senescence phenotype. Senescence in P. anserina, which is maternally inherited and associated with the excision and amplification of specific mitochondrial plasmids, occurs when this species is grown on solid medium. In two independent experiments no evidence of senescence was observed as mycelia were serially passaged in liquid culture. Further, when separable mycelial masses, termed puff balls, from the liquid cultures were plated on solid medium, a significant increase in their average longevity was observed. The apparent immortality of P. anserina in liquid culture was not dependent upon mitochondrial DNA rearrangements, nor was it affected by the presence of a previously described senescence plasmid, alpha senDNA. Evidence was obtained indicating that growth in liquid culture exerts selective pressure to maintain the wild-type mitochondrial genome.     

Promotion of the release of 11-cis-retinal from cultured retinal pigment epithelium by interphotoreceptor retinoid-binding protein.

This study investigates whether the interphotoreceptor retinoid-binding protein (IRBP) is necessary for the release of 11-cis-retinaldehyde (RAL) or if the retinoid is constitutively released from the retinal pigment epithelium (RPE) following synthesis. The strategic location of IRBP in the interphotoreceptor matrix (IPM) and its retinoid-binding ability make it a candidate for a role in 11-cis-RAL release. Fetal bovine RPE cells were grown in permeable chambers, and their apical surfaces were incubated with medium containing either apo-IRBP, the apo form of cellular retinaldehyde-binding protein (CRALBP), the apo form of serum retinol-binding protein (RBP), or bovine serum albumin (BSA) or with medium devoid of binding proteins. [3H]-all-trans-Retinol (ROL) was delivered to the basal surface of the cells by RBP. High-performance liquid chromatography demonstrated that [3H]-11-cis-RAL was optimally released into the apical medium when apo-IRBP was present. The most surprising result was the diminished level of [3H]-11-cis-RAL when apo-CRALBP was in the apical medium. Circular dichroism demonstrated that CRALBP had not been denatured by the photobleaching required for endogenous ligand removal. Therefore, apo-CRALBP should have been able to bind [3H]-11-cis-RAL if it was constitutively released into the apical medium. In addition, when proteins other than apo-IRBP were present, or if the cells were incubated with medium alone, the observed decrease in apical [3H]-11-cis-RAL was concomitant with a buildup of intracellular [3H]-all-trans-retinyl palmitate and [3H]-all-trans-ROL in the basal culture medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and purification of canadaphore, a siderophore produced by Helminthosporium carbonum

A new siderophore was isolated and purified from the spent growth medium of the fungus Helminthosporium carbonum by solvent extraction and reverse phase high pressure liquid chromatography. This new molecule has been assigned the name Canadaphore. Canadaphore was detected in culture filtrates after 15 days of growth and production was maximal after growth of H. carbonum to maximal stationary phase in modified Fries basal medium. Production of Canadaphore was completely suppressed when the organism was grown in medium supplemented with iron. Mass spectral analysis yielded a molecular mass of 680 Daltons for the iron-Canadaphore complex and 627 Daltons for the iron-free molecule. Spectroscopic analysis indicates that Canadaphore is a siderophore of the hydroxamate type.     

Vessels of the heart and lung in some experimental defects]

Under study were changes of intraorganic blood vessels of the heart and lungs in some experimental defects (open arterial defect, coarctation of the aorta, simultaneous existence of these two defects, stenosis of the pulmonary trunk, defect of the interatrial septum, triad of Fallot, syndrom of Lutembachet). Morphological data correlated with blood pressure in the pulmonary circulation and cardiac chambers. The complex of compensatory-adaptational mechanisms consisting of comparatively active and passive zones is formed in the heart and lungs. In most cases the changes develop in the vessels already existing. In hypertrophy of the myocardium when there is hypertension and hypervolemia in coronary vessels, sinusoids perform the function of blood reservoir, to a certain degree balancing the blood pressure, and luminar ducts relieve the muscle from excessive blood. The changes in the vascular system of the lung are directly dependent upon the pressure in the pulmonary circulation and the duration of observation. The closing arteries are the most active link in the chain of compensatory-adaptational mechanisms.     

Concentration polarization phenomenon in the case of mechanical pressure difference on the membrane

We analyzed the transport of KCl solutions through the bacterial cellulose membrane and concentration boundary layers (CBLs) near membrane with pressure differences on the membrane. The membrane was located in horizontal-plane between two chambers with different KCL solutions. The membrane was located in horizontal-plane between two chambers with different KCL solutions. As results from the elaborated model, gradient of KCL concentration in CBLs is maximal at membrane surfaces in the case when pressure difference on the membrane equals zero. The amplitude of this maximum decreases with time of CBLs buildup. Application of mechanical pressure gradient in the direction of gradient of osmotic pressure on the membrane causes a shift of this maximum into the chamber with lower concentration. In turn, application of mechanical pressure gradient directed opposite to the gradient of osmotic pressure causes the appearance of maximum of concentration gradient in chamber with higher concentration. Besides, the increase of time of CBLs buildup entails a decrease of peak height and shift of this peak further from the membrane. Similar behavior is observed for distribution of energy dissipation in CBLs but for pressure difference on the membrane equal to zero the maximum of energy dissipation is observed in the chamber with lower concentration. We also measured time characteristics of voltage in the membrane system with greater KCl concentrations over the membrane. We can state that mechanical pressure difference on the membrane can suppress or strengthen hydrodynamic instabilities visible as pulsations of measured voltage. Additionally, time of appearance of voltage pulsations, its amplitude, and frequency depend on mechanical pressure differences on the membrane and initial quotient of KCl concentrations in chambers.     

Characterization of serum, liver, and intestinal sialyltransferases from rats treated with colchicine

A modified high pressure liquid chromatographic method using lactose (Gal beta 1----4Glc) as an exogenous acceptor has been used to characterize the sialyltransferases known to increase in the serum of colchicine-treated rats. The results show a 10-fold increase of Gal beta 1----4GlcNAc alpha 2----6 sialyltransferase (alpha 2----6 ST), whereas the Gal beta 1----3GlcNAc alpha 2----3 sialyltransferase showed only 1.6-fold increase in the serum after 17 h of colchicine treatment. The sialyltransferase activity in serum using exogenous desialylated, alpha 1-acid glycoprotein as acceptor also showed an eightfold increase. In liver homogenate and Golgi membrane, the sialyltransferase activity when assayed with desialylated alpha 1-acid glycoprotein as acceptor showed a slight decrease after 4 h, but returned to normal level after 17 h. A similar trend was seen when the two transferases were assayed with lactose as acceptor. The antiserum to rat alpha 2----6 ST inhibited the sialyltransferase activity in serum, liver, and jejunal incubation medium. Jejunal sections from rats treated with colchicine for 4 h in presence of heated serum showed a decrease of sialyltransferase, with consequent increase of the alpha 2----6 ST enzyme activity in the medium. This result suggests that intestinal tissue could be a source of increased serum enzyme activity in colchicine treatment.     

Taxonomy of the marine,luminous bacteria

Summary One hundred and seventy-three strains of marine, luminous bacteria isolated from sea water, surfaces and intestines of fish, as well as from the luminous organs of fish and squid were submitted to an extensive phenotypic characterization. A numerical analysis of the results grouped these strains into four clusters which were formed on the basis of overall phenotypic similarity. One cluster, which was given the designationBeneckea harveyi, consisted of strains which had a moles% GC content in their DNAs of 46.5±1.3 and a single, sheathed, polar flagellum when grown in liquid medium. Most of these strains had unsheathed, peritrichous flagella in addition to the sheathed, polar flagellum when grown on solid medium. The two phenotypically similar clusters which were assigned the species designationsPhotobacterium phosphoreum andP. mandapamensis consisted of strains which had 1–3 unsheathed, polar flagella and moles % GC contents in their DNAs of 41.5±0.7 and 42.9±0.5, respectively. The cluster designatedP. fischeri contained strains having 2–8 sheathed, polar flagella and a moles % GC content of 39.8±1.1. These four species could be further distinguished on the basis of a number of nutritional properties as well as other phenotypic traits. The assignment of the luminous, marine bacteria to four species was supported by differences in the properties of the luminous system as well as differences in the pattern of regulation of spartokinase activity which are discussed. The speciesB. harveyi was found to be phenotypically similar to a number of previously characterized, non-luminous strains ofBeneckea which should probably be assigned to this species.Non-Standard Abbreviations ASW artificial sea water - ATCC American Type Culture Collection - BM basal medium - BMA basal medium agar - GC guanine plus cytosine - LA luminous medium agar - LB luminous medium broth - MA Difco Marine Agar - NCMB National Collection of Marine Bacteria - PHB poly--hydroxybutyrate - S similarity coefficient - YEB yeast extract broth This paper is part of a dissertation submitted by the senior author to the Graduate Division of the University of Hawaii in partial fulfillment of the requirements for the Ph.D. Degree in Microbiology     

Detection of salinity by the lobster, Homarus americanus.

Changes in the heart rates of lobsters (Homarus americanus) were used as an indicator that the animals were capable of sensing a reduction in the salinity of the ambient seawater. The typical response to a gradual (1 to 2 ppt/min) reduction in salinity consisted of a rapid increase in heart rate at a mean threshold of 26.6 +/- 0.7 ppt, followed by a reduction in heart rate when the salinity reached 22.1 +/- 0.5 ppt. Animals with lesioned cardioregulatory nerves did not exhibit a cardiac response to changes in salinity. A cardiac response was elicited from lobsters exposed to isotonic chloride-free salines but not to isotonic sodium-, magnesium- or calcium-free salines. There was little change in the blood osmolarity of lobsters when bradycardia occurred, suggesting that the receptors involved are external. Furthermore, lobsters without antennae, antennules, or legs showed typical cardiac responses to low salinity, indicating the receptors are not located in these areas. Lobsters exposed to reductions in the salinity of the ambient seawater while both branchial chambers were perfused with full-strength seawater did not display a cardiac response until the external salinity reached 21.6 +/- 1.8 ppt. In contrast, when their branchial chambers were exposed to reductions in salinity while the external salinity was maintained at normal levels, changes in heart rate were rapidly elicited in response to very small reductions in salinity (down to 29.5 +/- 0.9 ppt in the branchial chamber and 31.5 +/- 0.3 ppt externally). We conclude that the primary receptors responsible for detecting reductions in salinity in H. americanus are located within or near the branchial chambers and are primarily sensitive to chloride ions.     

Lactose variability of Escherichia coli in thermally stressed reactor effluent waters.

Lactose-utilizing and nalidixic acid-resistant populations of Escherichia coli, having an optimum growth temperature of 37 degrees C, were placed in modified diffusion chambers. The chambers were submerged in the epilimnion and hypolimnion of a 1,100-hectare lake (Par Pond) which receives cooling water from a nuclear production reactor. Control chambers were placed in a deep-water reservoir and a Flowing-Streams Laboratory, both of which had comparable temperatures to Par Pond. The populations of E. coli were sampled regularly for up to 3 weeks. Viability of the bacteria was determined by dilution plating to nutrient agar followed by replicate plating onto selective medium to determine lactose utilization and nalidixic acid sensitivity. Initial populations of E. coli were lactose positive but changed to lactose negative in Par Pond when the reactor was operating (i.e., cooling water from the heat exchangers was being discharged to the lake). This alteration occurred most rapidly in the chambers closest to the cooling-water discharge point. Such changes did not occur in a deep-water reservoir, in Par Pond when the reactor was not operating, or in the Flowing-Streams Laboratory. The nalidixic acid-resistant characteristic remained stable regardless of the chambers' placement or reactor operations. Although the reasons for such alterations are unclear, it appears that lactose-negative populations of E. coli are selected for in these reactor effluent waters. The loss of the lactose characteristic prevents the recognition and identification of E. coli in this cooling lake (when the reactor is operating) and may prevent the assessment of water quality based on coliform recognition.     

The Effects of Agitated Liquid Medium on in vitro Cultures of Hevea brasiliensis

The initiation and prolonged growth of callus, from stem explants of young plants of Hevea brasilienies on solid medium yielded a heterogeneous callus, with areas which are the result of compact growth interspersed with brown necrotic tissue and soft white tissue formations. Subculturing this callus (O callus) to agitated liquid medium and returning it to solid medium resulted in the production of a homogeneous friable and rapidly growing callus (Rl callus) The two established lines O and Rl have remained stable over one year in culture and differ in gross morphology, anatomy, growth and auxin content. Both were maintained on Murashige and Skoog's medium, with 2 mg/1 2,4-D and 0.5 mg/I kinetin. R 1 but not O showed enhanced growth at the lower 2,4-D level of 0.2 mg/l: both lines failed to continue growing when 2,4-D was omitted. It is suggested that the changes resulting from subculture in agitated liquid medium are related to those undergone by callus cultures which become habituated. Thus the Rl callus line is regarded as partially habituated. Subculture in agitated liquid medium also resulted in the production of large numberr of polyploid cells but these did not persist over the long periods of subsequent growth on agar medium, Enhanced auxin production by the establihed Rl callus line was thus observed in the absence of a detectable level of polyploidy.     

Improved Microscopy of Mycoplasma In Vitro

Techniques were developed for continuous microscopic observation of mycoplasmata growing in vitro in Rose chambers by using an inverted phase microscope. The methods permitted direct microscopic observation of undisturbed growth of mycoplasmata in liquid medium. Inocula of mycoplasmata were passed through 0.22-mum filters before culture to provide a suspension of discrete particles. The sequential growth of Mycoplasma pneumoniae was followed from points or single straight lines, with development of branching, a net-like confluence of filaments, large bodies occurring in the center of developing colonies, and finally coccoid forms. Other species of Mycoplasma which did not attach as readily to glass could be observed also by inverted phase microscopy. Umbonation of colonies (a "friedegg" appearance) occurred in liquid medium, indicating that this appearance was not due simply to interaction with the agar medium, but may reflect a qualitative difference in growth patterns between center and periphery. For growth on solid medium, direct observation of colonies in uncovered plates of agar medium was made by using inverted phase microscopy. This was found helpful in detecting small colonies and in observing relationships between colonies.     

Hormonal and purinergic stimulation of bicarbonate secretion in oviducts of rhesus monkey

Because an increase in the HCO(3)(-) concentration of oviductal liquid at midcycle is believed to markedly enhance fertility, we have studied active secretion of HCO(3)(-) across highly differentiated cultures of monkey oviductal epithelium. Cultured cell sheets were mounted in Ussing chambers and bathed in medium containing 25 mM HCO(3)(-). Purinergic agents potently stimulated short-circuit current (I(sc)) with an initial transient response declining within approximately 2 min to a sustained response. The potency sequence of ATP approximately UTP > ADP > AMP suggested that the I(sc) response was mediated mainly by P2Y(2) receptors. Acetazolamide, an inhibitor of carbonic anhydrase, had little or no effect on baseline I(sc) or the transient response to ATP but abolished the sustained response to ATP. Similar results were obtained on sheets of native epithelium. In pH-stat experiments, the abluminal medium of cell cultures was bathed in HCO(3)(-)-CO(2) medium, and the pH of the unbuffered luminal medium was maintained at approximately 7.4 by addition of strong acid or base. ATP stimulated base secretion, and this was inhibited by acetazolamide. Furthermore, these changes in secretion of base were in good quantitative agreement with the I(sc) responses. When phenol red (an estrogen) was removed from the culture medium, ATP-dependent HCO(3)(-) secretion was markedly reduced but could be restored by treatment with estradiol. Estrogens also markedly increased ciliation of the cultures. These results suggest that the midcycle increase in the HCO(3)(-) concentration of oviductal liquid may be mediated by the effects of estradiol on purinergic pathways or on ATP secretion.     

A two-compartment cell entrapment bioreactor with three different holding times for cells,high and low molecular weight compounds

A new bioreactor for animal cell cultivation employs two compartments for cells and medium respectively. The two chambers are separated by an ultrafiltration membrane. Cells and solution of collagen or collagen/chitosan mixture were loaded to the cell chamber and were allowed to form gel inside. Contraction of the cell-laden gel occurred subsequently to create a new zone in the cell chamber. In such a bioreactor cells are retained in the reactor, the high molecular product(s) accumulate in the cell chamber, while the small molecular weight nutrients and metabolites are replenished and removed from the medium chamber. By adjusting the flow rates for cell and medium chambers, the resident time for cells, high and low molecular weight components of the system can be manipulated separately. The new bioreactor, in both flat-bed and hollow-fiber configurations, was used to cultivate recombinant human cell, 293, for Protein C production over 60 to 90 days.