Determination of the density of cells from sedimentation studies at 1G

To obtain a possible correlation between cell density and cell size, the size of individual cells was measured under the microscope and their sedimentation velocity was measured; the density is obtained with Stoke's Law. Specifically, HeLa cells were sedimented in Joklik's medium at 30°C in a vertical glass tube with 2 mm×2 mm horizontal opening and cells observed with a horizontally aligned microscope. The overall mean density difference of HeLa cells at 30°C was 0.0316+–0.0044 g/cm³, resulting in a density of 1.0357 g/cm³ (with a density of 1.0040 g/cm³ for Joklik's medium at 30°C). Six size fractions had densities which were essentially the same within the errors of the mean densities of the fractions (from 0.0081 to 0.0202 g/cm³). The considerably varying deviations of individual densities from the mean suggested superimposed phenomena (see also Table I for microspheres of precise size). Careful observation in balancing countercurrent flow revealed microconvection over 5 to 15 m regions, most likely based on small thermal differences in the horizontal plane.     

Fluid flow through a high cell density fluidized‐bed during centrifugal bioreactor culture

An increasing demand for products such as tissues, proteins, and antibodies from mammalian cell suspension cultures is driving interest in increasing production through high‐cell density bioreactors. The centrifugal bioreactor (CCBR) retains cells by balancing settling forces with surface drag forces due to medium throughput and is capable of maintaining cell densities above 10⁸ cells/mL. This article builds on a previous study where the fluid mechanics of an empty CCBR were investigated showing fluid flow is nonuniform and dominated by Coriolis forces, raising concerns about nutrient and cell distribution. In this article, we demonstrate that the previously reported Coriolis forces are still present in the CCBR, but masked by the presence of cells. Experimental dye injection observations during culture of 15 μm hybridoma cells show a continual uniform darkening of the cell bed, indicating the region of the reactor containing cells is well mixed. Simulation results also indicate the cell bed is well mixed during culture of mammalian cells ranging in size from 10 to 20 μm. However, simulations also allow for a slight concentration gradient to be identified and attributed to Coriolis forces. Experimental results show cell density increases from 0.16 to 0.26 when centrifugal force is doubled by increasing RPM from 650 to 920 at a constant inlet velocity of 6.5 cm/s; an effect also observed in the simulation. Results presented in this article indicate cells maintained in the CCBR behave as a high‐density fluidized bed of cells providing a homogeneous environment to ensure optimal growth conditions. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

TIP CELL GROWTH AND THE FREQUENCY AND DISTRIBUTION OF CELLULOSE MICROFIBRIL-SYNTHESIZING COMPLEXES IN THE PLASMA MEMBRANE OF APICAL SHOOT CELLS OF THE RED ALGA PORPHYRA YEZOENSIS1

The supramolecular organization of the plasma membrane of apical cells in shoot filaments of the marine red alga Porphyra yezoensis Ueda (conchocelis stage) was studied in replicas of rapidly frozen and fractured cells. The protoplasmic fracture (PF) face of the plasma membrane exhibited both randomly distributed single particles (with a mean diameter of 9.2 ± 0.2 nm) and distinct linear cellulose microfibril-synthesizing terminal complexes (TCs) consisting of two or three rows of linearly arranged particles (average diameter of TC particles 9.4 plusmn; 0.3 nm). The density of the single particles of the PF face of the plasma membrane was 3000 μm^?2, whereas that of the exoplasmic fracture face was 325 μm^?2. TCs were observed only on the PF face. The highest density of TCs was at the apex of the cell (mean density 23.0 plusmn; 7.4 TCs μm^?2 within 5 μm from the tip) and decreased rapidly from the apex to the more basal regions of the cell, dropping to near zero at 20 μm. The number of particle subunits of TCs per μm² of the plasma membrane also decreased from the tip to the basal regions following the same gradient as that of the TC density. The length of TCs increased gradually from the tip (mean length 46.0 plusmn; 1.4 nm in the area at 0–5 μm from the tip) to the cell base (mean length 60.0 plusmn; 7.0 μm in the area at 15–20 μm). In the very tip region (0–4 μm from the apex), randomly distributed TCs but no microfibril imprints were observed, while in the region 4–9 μm from the tip microfibril imprints and TCs, both randomly distributed, occurred. Many TCs involved in the synthesis of cellulose microfibrils were associated with the ends of microfibril imprints. Our results indicate that TCs are involved in the biosynthesis, assembly, and orientation of cellulose microfibrils and that the frequency and distribution of TCs reflect tip growth (polar growth) in the apical shoot cell of Porphyra yezoensis. Polar distribution of linear TCs as “cellulose synthase” complexes within the plasma membrane of a tip cell was recorded for the first time in plants.     

OPOSSUM KIDNEY CELLS TAKE UP L-DOPA THROUGH AN ORGANIC CATION POTENTIAL-DEPENDENT AND PROTON-INDEPENDENT TRANSPORTER

The present work was aimed at studying the kinetics and nature of the l-DOPA transporter in opossum kidney (OK) cells. Saturation experiments were performed in OK cells incubated for 6 min with increasing concentrations of l-DOPA (10 to 2500 μm); non-linear analysis of the saturation curve revealed for l-DOPA aK_mof 129 μm (114, 145) and aV_maxof 30.0±0.4 nmol mg protein^?16 min^?1The uptake of l-DOPA (250 μm) was inhibited in a concentration-dependent manner by cyanine 863, an organic cation inhibitor, with aK_ivalue of 638 (430, 947) μmthe organic anion inhibitor 4,4′-diisothiocyanostilbene-2,2′-disulphonic acid (DIDS), was devoid of effect upon the uptake of l-DOPA. The uptake of l-DOPA (250 μm) was significantly (P<0.02) decreased (25% reduction) when cells were incubated in the presence of 137 mm K⁺plus 5 mm Na⁺when compared with the control condition (137 mm Na⁺plus 5 mm K⁺); substitution of NaCl by choline chloride (137 mm) did not affect l-DOPA uptake. Similarly, inwardly or outwardly directed proton gradients of 0.5 pH units (7.9, 7.4, 6.9, 6.4 and 5.9) were found not to change l-DOPA uptake. In conclusion, the l-DOPA uptake system in OK cells has the characteristics of an organic cation potential-dependent and proton-independent transporter.     

Nanomapping of CD1d–glycolipid complexes on THP1 cells by using simultaneous topography and recognition imaging

CD1d molecule, a monomorphic major histocompatibility complex class I‐like molecule, presents different types of glycolipids to invariant natural killer T (iNKT) cells that play an important role in immunity to infection and tumors, as well as in regulating autoimmunity. Here, we present simultaneous topography and recognition imaging (TREC) analysis to detect density, distribution and localization of single CD1d molecules on THP1 cells that were loaded with different glycolipids. TREC was conducted using magnetically coated atomic force microscopy tips functionalized with a biotinylated iNKT cell receptor (TCR). The recognition map revealed binding sites visible as dark spots, resulting from oscillation amplitude reduction during specific binding between iNKT TCR and the CD1d–glycolipid complex. THP1 cells were pulsed with three different glycolipids (α‐GalCer, C20 and OCH12) for 4 and 16 hr. Whereas CD1d–α‐GalCer and CD1d–C20:2 complexes on cellular membrane formed smaller microdomains up to ~10 000 nm² (dimension area), OCH12 loaded CD1d complexes presented larger clusters with a dimension up to ~30 000 nm². Moreover, the smallest size of recognition spots was about 25 nm, corresponding to a single CD1d binding site. TREC successfully revealed the distribution and localization of CD1d–glycolipid complexes on THP1 cell with single molecule resolution under physiological conditions. Copyright © 2013 John Wiley & Sons, Ltd.     

High cell density cultivation of human leukemia T cells (Jurkat cells) in semipermeable polyelectrolyte microcapsules

The ex vivo expansion of human T cells is of considerable scientific and medical interest. Currently, this requires the addition of massive amounts of stimuli. Here, human leukemia T cells (Jurkat cells) were used as model cells to demonstrate the in vitro expansion of T cells in the absence of added stimuli after encapsulation in semipermeable sodium cellulose sulfate/poly(diallyldimethyl) ammonium chloride polyelectrolyte membrane capsules (molecular weight cutoff <10 kDa, average diameter ca. 800 μm). For comparison, free and encapsulated cells were cultivated in standard T‐flasks and spinner bottles (both 50 mL culture medium) as well as in hanging drops (35 μL, only nonencapsulated cells). Encapsulation led to a significantly higher specific growth rate, a prolonged exponential growth phase together with a reduced tendency for apoptosis, as evidenced by shifts in the cell cycle distribution toward the S and G2/M phases together with a reduced percentage of cells in the sub‐G0/G1 phase. As a consequence, very high cell densities (>140×10⁶ cells/mL_capsule) were obtained in the capsules, particularly for the spinner cultivations. No evidence for nonspecific activation/stimulation, that is IL‐2 and CD25 expression, was found, while specific stimulation by phorbol‐12‐myristate‐13‐acetate/ionomycin was still possible. Since Jurkat cells commonly serve as model cells for primary T lymphocytes, the proposed method may present a strategy for high‐density proliferation of primary human T lymphocytes.     

Retinal ganglion cell topography and visual acuity of the sleepy lizard (<Emphasis Type="Italic">Tiliqua rugosa</Emphasis>)

The spatial distribution of retinal ganglion cells provides valuable insight into the importance species place on observing objects in specific regions of their visual field with higher spatial resolving power. We estimate the total number, distribution and peak density of ganglion cells in retinal wholemounts of the sleepy lizard, Tiliqua rugosa, a scincid lizard endemic to southern Australia. Ganglion cells were readily discernable from amacrine cells by their size and shape, prominent nuclei and the accumulation of Nissl-positive substances in their cytoplasm. A total of 1,654,200 (±59,400) presumed ganglion cells were estimated throughout the retina, distributed irregularly and forming a loose horizontal streak of high cell density peaking at 15,500 cells per mm². With a post nodal distance of 6.25 mm, we calculate an upper limit of visual acuity of 6.8 c/deg.     

Measurement of the size distribution of human erythrocytes by a sedimentation method

The distribution of sedimentation velocities was determined, by a photoelectric method, for human erythrocytes at low concentrations in Ringer solution. The light absorption at 414 nm was measured, as a function of time, 10 mm below the top of the column. From the frequency distribution of cell velocities that of R_s √ρ-σ was found; R_s being the Stokes' radius, ρ the cell density and σ the density of the solution. Cell density was measured by the phthalate method and the mean Stokes' radius was found to be 2.58 μm. The size distributions showed some skewness but were in good general agreement with those measured by Celloscope counter, and with reported measurements from photomicrographs of cells in hanging drop suspensions. The skewness was much less than that encountered with electric sensing zone instruments (e.g. Celloscope) and the sedimentation method, being based on entirely different premises, provides an important check on such data. The skewness is due to a bias in the orientation of human erythrocytes during sedimentation. This bias may be a characteristic of biconcave cells; it could be absent in many species and reliable measurements of size distribution would then be obtained.     

Effects and mechanisms of ghrelin on cardiac microvascular endothelial cells in rats

Ghrelin is thought to directly exert a protective effect on the cardiovascular system, specifically by promoting vascular endothelial cell function. Our study demonstrates the ability of ghrelin to promote rat CMEC (cardiac microvascular endothelial cell) proliferation, migration and NO (nitric oxide) secretion. CMECs were isolated from left ventricle of adult male Sprague—Dawley rat by enzyme digestion and maintained in endothelial cell medium. Dil‐ac‐LDL (1,1′‐dioctadecyl‐3,3,3′,3′‐ tetramethylindocarbocyanine‐labelled acetylated low‐density lipoprotein) intake assays were used to identify CMECs. Cells were split into five groups and treated with varying concentrations of ghrelin as follows: one control non‐treated group; three ghrelin dosage groups (1×10^?9, 1×10^?8, 1×10^?7 mol/l) and one ghrelin+PI3K inhibitor group (1×10^?7 mol/l ghrelin+20 μmol/l LY294002). After 24 h treatment, cell proliferation capability was measured by MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H‐tetrazolium bromide] assay and Western blot for PCNA (proliferating cell nuclear antigen) protein expression. Migration of CMECs was detected by transwell assays, and NO secretion of CMECs was measured via nitrate reduction. Protein expression of AKT and phosphorylated AKT in CMECs was measured by Western blot after exposure to various concentrations of ghrelin and the PI3K inhibitor LY294002. Our results indicate that ghrelin significantly enhanced cell growth at concentrations of 10^?8 mol/l (0.271±0.041 compared with 0.199±0.021, P=0.03) and 10^?7 mol/l (0.296±0.039 compared with 0.199±0.021, P<0.01). However, addition of the PI3K/AKT inhibitor LY294002 inhibited the ghrelin‐mediated enhancement in cell proliferation (0.227±0.042 compared with 0.199±0.021, P=0.15). At a concentration between 10^?8 and 10^?7 mol/l, ghrelin caused a significant increase in the number of migrated cells compared with the control group (126±9 compared with 98±7, P=0.02; 142±6 compared with 98±7, P<0.01), whereas no such change could be observed in the presence of 20 μmol/l of the PI3K/Akt inhibitor LY294002 (103±7 compared with 98±7, P=0.32). Ghrelin treatment significantly enhanced NO production in a dose‐dependent fashion compared with the untreated control group [(39.93±2.12) μmol/l compared with (30.27±2.71) μmol/l, P=0.02; (56.80±1.98) μmol/l compared with (30.27±2.71) μmol/l, P<0.01]. However, pretreatment with 20 μmol/l LY294002 inhibited the ghrelin‐stimulated increase in NO secretion [(28.97±1.64) μmol/l compared with (30.27±2.71) μmol/l, P=0.37]. In summary, we have found that ghrelin treatment promotes the proliferation, migration and NO secretion of CMECs through activation of PI3K/AKT signalling pathway.     

The role of glutathione in rat adipocyte pentose phosphate cycle activity

An assay for reduced and oxidized glutathione was adapted to isolated rat epididymal adipocytes in order to correlate pentose phosphate cycle activity and glutathione metabolism. In collagenase-digested adipocytes the [GSH/GSSG] molar ratio was in excess of 100. Cells incubated for 1 hr with low glucose concentrations (0.28–0.55 mm) had higher GSH contents (3.2 μg/10⁶ cells) than in the absence of glucose (2.3 μg/10⁶ cells). The glutathione oxidant diamide caused a dose-related decrease in intracellular GSH, an increase in GSSG released into the medium, but no detectable change in the low intracellular GSSG content. The intracellular content of GSH and amount of GSSG released into the medium were therefore taken to reflect the glutathione status of the adipocytes most closely. Addition of H₂O₂ to a concentration of 60 μm to adipocytes caused to decline within 5 min in GSH content, which was less severe and more rapid to recover in the presence of 1.1 mm glucose, suggesting that the concomitant stimulation of glucose C-1 oxidation induced by the peroxide in the presence of glucose provided NADPH for regeneration of GSH. Further evidence for tight coupling between adipocyte [GSH/GSSG] ratios and pentose phosphate cycle activity was that (i) lowering intracellular GSH to 35–60% of control values by agents as diverse in action as t-butyl hydroperoxide, diamide, or the sulfhydryl blocker N-ethylmaleimide resulted in optimal stimulation of glucose C-1 oxidation and fractional pentose phosphate cycle activity, and (ii) incubating adipocytes directly with 2.5 mm GSSG resulted in a slight increase in glucose C-1 oxidation and when 0.5 mm NADP⁺ was also added a synergistic effect on pentose phosphate cycle activity was found. On the other hand, electron acceptors such as methylene blue did not lower cellular GSH content, but did stimulate the pentose phosphate cycle, confirming a site of action independent of glutathione metabolism. The results show that (i) glucose metabolism by the pentose phosphate cycle contributes to regeneration of GSH and that (ii) glutathione metabolism either directly or via coupled changes in [NADPH/NADP⁺] ratios may play a significant role in short-term control of the pentose phosphate cycle.     

Characterization of Vero cell growth and death in bioreactor with serum-containing and serum-free media

The density of viable cells in a culture results from a balance between cell proliferation and cell death. The aim of this study was to characterize and compare these two phenomena in Vero cell cultures in one serum containing medium (ScA) and one serum free medium (SfB) in bioreactors. Cell growth was evaluated by cell counting(after crystal violet staining) and cell cycle analysis. Necrosis and apoptosis were characterized and quantified by measuring the release of LDH, trypan blue exclusion,annex in V-FITC/PI staining and TUNEL assay. ScA supported a higher maximal viable-cell density(2.3 × 10⁶ vs. 1.8 × 10⁶ cells ml^-1). However, cell cycle analysis showed that cell division was more active in SfB than in ScA. LDH release in the supernatant increased much earlier in SfB than in ScA (one vs. five days), but trypan blue counts showed no apparent difference in the viability of the cultures. Apoptosis, evidenced by annexin V-FITC/PI staining, could be detected in the population of suspension cells detached from microcarriers, but not among adherent cells; positivity of the TUNEL assay occurred later than that of the annexin V-FITC/PI staining. Our data indicate that the lower cell yield in SfB,compared with that in ScA, results from a higher cell death rate. Apparently, cells die from apoptosis followed by secondary necrosis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Picoplankton in Lake Maggiore,Italy

In 1992 we examined the morphological characteristics and space-time distribution of picoplankton cells in Lake Maggiore, a subalpine lake in which oligotrophication is in progress. We measured by image analyser the biovolume of autotrophic (APP), eukaryotic and prokaryotic. and heterotrophic (HPP) picoplankton. Among the APP < 2μm the yellow fluoresceing are the dominating cells in the euphoric zone. The red cells, mainly eukaryotic, on average are only 11% of the total abundance of cells < 5μm. The APP cell numbers range from 9.5 × 10³ cells ml⁻¹ to 1.3 × 10⁵ cells ml⁻¹ (average: 5 × 10⁴ cells ml⁻¹). Their mean biovolume shows a minimum value of 7.8mm³ m⁻³ in March and a maximum of 186.3mm³ m-³ in September. The contribution of biovolume of yellow cells to total phytoplankton biovolume varies between 0.3% and 27%. suggesting that picocyanobacteria, at this stage of lake recovery, are not yet a dominant component. The HPP cell density is two order of magnitude higher than the APP with a mean value of 2.6 × 10⁶ cells ml⁻¹. APP mean cell size fluctuates from a minimum of 0.5 μm to a maximum of 1.4urn (0.26–1.69μm³ volume), while HPP range from 0.4 to 0.7 um (0.07–0.57 μm³ volume), making it easier to distinguish them on a dimensional basis for most of the year. During the period of thermal stratification, a peak in abundance was noted in the central part of the metalimnion at depths receiving less than 10% of surface irradiance. The total picoplanktonic carbon fraction (APP and HPP) varied from 38 to 384 μgC 1⁻¹ with a mean value of 133μgC 1⁻¹ which represents 42% of POC collected on GF/C filters. Most of the picoplankton carbon is made up of HPP cells (34% of the total POC).     

Characterization of the methotrexate transport defect in a resistant L1210 lymphoma cell line

L1210/R81 lymphoma cells are resistant to methotrexate (MTX) by virtue of a 35-fold elevation in dihydrofolate reductase and an inability to transport the folate antagonist drug effectively. In a phosphate-containing buffer there was little or no influx into the resistant cells at either 1 or 50 μm MTX. Replacement of this buffer with a 4-(2-hydroxyethyl)-1-piperazine-N′-2-ethanesulfonic acid-Mg²⁺ system resulted in an apparent influx of MTX into the resistant cells. Under these conditions, L1210/R81 cells achieved an apparent steady state at an extracellular MTX concentration of 50 μm. The apparent steady-state level of 5 nmol $\text{[}{}^3\text{H]MTX}\text{10}{}^9$ cells was well below the intracellular level of dihydrofolate reductase (45 nmol/10⁹ cells). Efflux experiments at the apparent steady state indicated that 60% of the MTX was very rapidly removed from the cells by washing. Over the range of the experiment a further 20% of the MTX effluxed more slowly ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 12}\text{min}$). The apparent influx into the resistant cells at 5 μm MTX was inhibited 13% by sodium azide (100 μm) and initially stimulated, then inhibited, by p-chloromercuriphenylsulfonic acid (100 μm). 5-Methyltetrahydrofolate (100 μm) had little effect on the process while aminopterin (100 μm) was inhibitory (68%). K_t and V values of 2 × 10^?5m and 0.31 nmol $\text{[}{}^3\text{H]MTX}\text{10}{}^9$ cells/min, respectively, were determined for the apparent influx in $\text{L}\text{1210}\text{R}\text{81}$ cells. Comparison of apparent MTX influx in the resistant cells with MTX transport in the sensitive cells indicates profound differences in the two processes. The evidence suggests that the apparent influx in the former cell line may consist of MTX binding to the cell membrane together with a small degree of MTX influx into the intracellular compartment.     

Comparative effects of adenine analogs upon metabolic cooperation between Chinese hamster cells with different levels of adenine phosphoribosyltransferase activity

Colony formation by variant Chinese hamster cells highly resistant to adenine analogs and deficient in adenine phosphoribosyltransferase (APRT) activity was measured after co-cultivation with APRT⁺, CHO-K1 cells in medium containing one of three different adenine analogs. Depending upon the density of APRT⁺ cells and the specific adenine analog, large differences in the recovery of APRT^? colonies were observed. The particular adenine analog and APRT⁺ cell density were more significant factors in the recovery of APRT^? colonies than the concentration of the analog or the level of APRT activity. The number of wild-type cells (CHO-K1) required to inhibit formation of APRT^? colonies by 50% (mean lethal density; MLD₅₀) with 65 μg/ml 8-aza-adenine (AzA) as the selective drug was 8.0 × 10⁵ cells/100 mm dish (1.5 × 10⁴/cm²). With 100 μg/ml 2,6-diaminopurine (DAP) the MLD₅₀ for CHO-K1 was 4.0 × 10⁵ cells/100 mm dish (7.3 × 10³/cm²). The MLD₅₀ for CHO-K1 when the DAP concentration was decreased to 50 μg/ml was only slightly higher, 5 × 10⁵ cells/100 mm dish (9.1 × 10³/cm²). The most toxic effect was observed with 2-fluoroadenine (FA). The MLD₅₀ for CHO-K1 in 2 μg/ml FA was 4.5 × 10⁴ cells/100 mm dish (8.2 × 10²/cm²), a cell density which permits minimal direct contact between APRT⁺ and APRT^? cells. The toxic effects of FA on individually resistant, APRT^? cells were found to be mediated by metabolites released into the medium by dying APRT⁺ cells. This metabolite toxicity to APRT^? cells was also demonstrated in mixtures with cells having only 8% of wild-type APRT activity. The MLD₅₀ for these APRT⁺ (8%) cells in 2 μg/ml FA was 7.5 × 10⁴ cells/100 dish (1.4 × 10³/cm²), a small difference from the MLD₅₀ for cells with wild-type levels of APRT activity. The differences in the recovery of APRT^? colonies from mixtures with APRT⁺ cells in these three adenine analogs are critical to the design of procedures for the selection of APRT^? cells from populations of APRT⁺ cells and emphasize the importance of establishing the parameters of metabolic cooperation, not only in terms of cell density but also with regard to the particular selective agent, in any experiment designed to determine precise mutation rates or to test putative mutagens upon mammalian cells in culture.     

Deoxyadenosine toxicity in an adenosine deaminase-inhibited human CCRF-CEM T-lymphoblastoid cell line causes cell swelling

The human T-lymphoblastoid cell line CCRF-CEM, pre-treated with 2′-deoxycoformycin, was used as a model for adenosine deaminase deficiency to investigate how 2′-deoxyadenosine exerts its cytotoxic effects. Incubation of these cells with 1 μM or 5 μM deoxyadenosine for 24 and 48 h caused an increase of up to 50% in their modal cell volume as measured by a Coulter Size Distribution Analyzer and this increase in cell volume was accompanied by an increase in their fragility and deformability. The swelling of cells was concomitant with the phosphorylation of deoxyadenosine and its intracellular accumulation as dATP. There was no evidence of osmotic imbalance or of inhibition of the Na⁺/K⁺-dependent ATPase activity as the intracellular concentrations (and the intracellular: extracellular ratios)_of Na⁺, K⁺ and Ca²⁺ were essentially unchanged. Cytochalasin B (20 μM) also caused lymphoblasts to swell over a 6-h period and its effect on cell size was similar to that of either 1 μM or 5 μM deoxyadenosine over 24 or 48 h. Longer time-courses of incubation with cytochalasin B caused severe toxicity leading to the death and lysis of a significant proportion of the cells. Other drugs, such as colchicine, vincristine and vinblastine that are known to affect various components of the cytoskeleton also caused swelling of cells in a concentration- and time-dependent manner but there was no evidence that these effects were additive or synergistic with those of deoxyadenosine. Inhibition of DNA synthesis, either directly by aphidicolin or indirectly by hydroxyurea, was less cytotoxic than the effect caused by deoxyadenosine. We conclude that one of the toxic effects resulting from the excessive phosphorylation of deoxyadenosine and its accumulation as dATP in human T-lymphoblasts is not dependent on inhibition of DNA synthesis but may be caused by the disruption of the cytoskeleton in these cells.     

293 cell cycle synchronisation adenovirus vector production

As the market requirements for adenovirus vectors (AdV) increase, the maximisation of the virus titer per culture volume per unit time is a key requirement. However, despite the fact that 293 cells can grow up to 8 × 10⁶ cell/mL in simple batch mode operations, for optimal AdV infection a maximum cell density of 1 × 10⁶ cell/mL at infection time has usually been utilized due to the so called “cell density effect”. In addition, AdV titer appears to be dependent upon cell cycle phase at the time of infection. To evaluate the dependence of AdV production upon cell cycle phase, 293 cells were chemically synchronised at each phase of the cell cycle; a 2.6‐fold increase on AdV cell specific titer was obtained when the percentage of cells at the S phase of the cell cycle was increased from 36 to 47%; a mathematical equation was used to relate AdV cell specific productivities with cell synchronisation at the S phase using this data. To avoid the use of chemical inhibitors, a temperature shift strategy was also used for synchronisation at the S phase. S phase synchronisation was obtained by decreasing the culture temperature to 31°C during 67 h and restoring it to 37°C during 72 h. By using this strategy we were able to synchronise 57% of the population in the S phase of the cell cycle obtaining an increase of 7.3‐fold on AdV cell specific titer after infection. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Electron microscopic detection and in situ characterization of bacterial nanoforms in extreme biotopes

The morphology, ultrastructure, and quantity of bacterial nanoforms were studied in extreme biotopes: East Siberia permafrost soil (1–3 Ma old), petroleum-containing slimes (35 years old), and biofilms from subsurface oil pipelines. The morphology and ultrastructure of microbial cells in natural biotopes in situ were investigated by high-resolution transmission electron microscopy and various methods of sample preparation: ultrathin sectioning, cell replicas, and cryofractography. It was shown that the biotopes under study contained high numbers of bacterial nanoforms (29–43% of the total number of microorganisms) that could be assigned to ultramicrobacteria due to their size (diameter of ≤ 0.3 μm and volume of ≤ 0.014 μm³) and structural characteristics (the presence of the outer and cytoplasmic membranes, nucleoid, and cell wall, as well as their division patterns). Seven different morphostructural types of nanoforms of vegetative cells, as well as nanospores and cyst-like cells were described, potentially representing new species of ultramicrobacteria. In petroleum-containing slimes, a peculiar type of nanocells was discovered, gram-negative cells mostly 0.18–0.20 × 0.20–0.30 μm in size, forming in situ spherical aggregates (microcolonies) of dividing cells. The data obtained promoted the isolation of pure cultures of ultramicrobacteria from petroleum-containing slimes; they resembled the ultramicrobacterium observed in situ in their morphology and ultrastructure.     

Morphological and biochemical characterization of mineralizing primary cultures of avian growth plate chondrocytes: Evidence for cellular processing of Ca2+ and Pi prior to matrix mineralization

Advances in the culture of mineralizing growth plate chondrocytes provided an opportunity to study endochondral calcification under controlled conditions. Here we report that these cultures synthesize large amounts of proteins characteristically associated with mineralization: type II and X collagens, sulfated proteoglycans, alkaline phosphatase, and the bone-related proteins, osteonectin and osteopontin. Certain chondrocytes appeared to accumulate large amounts of Ca²⁺ and Pi during the mineralization process: laser confocal imaging revealed high levels of intracellular Ca²⁺ in their periphery and X-ray microanalytical mapping revealed the presence of many Ca²⁺- and Pi-rich cell surface structures ranging from filamentous processes 0.14 ± 0.02 μm by 0.5–2.0 μm, to spherical globules 0.70 ± 0.27 μm in diameter. Removal of organic matter with alkaline sodium hypochlorite revealed numerous deposits of globular (0.77 ± 0.19 μm) mineral (calcospherites) in the lacunae around these cells. The size and spatial distribution of these mineral deposits closely corresponded to the Ca²⁺-rich cell surface blebs. The globular mineral progressively transformed into clusters of crystallites. Taken with earlier studies, these findings indicate that cellular uptake of Ca²⁺ and Pi leads to formation of complexes of amorphous calcium phosphate, membrane lipids, and proteins that are released as cell surface blebs analogous to matrix vesicles. These structures initiate development of crystalline mineral. Thus, the current findings support the concept that the peripheral intracellular accumulation of Ca²⁺ and Pi is directly involved in endochondral calcification.     

Very high density of CHO cells in perfusion by ATF or TFF in WAVE bioreactor™. Part I. Effect of the cell density on the process

High cell density perfusion process of antibody producing CHO cells was developed in disposable WAVE Bioreactor? using external hollow fiber filter as cell separation device. Both “classical” tangential flow filtration (TFF) and alternating tangential flow system (ATF) equipment were used and compared. Consistency of both TFF‐ and ATF‐based cultures was shown at 20–35 × 10⁶ cells/mL density stabilized by cell bleeds. To minimize the nutrients deprivation and by‐product accumulation, a perfusion rate correlated to the cell density was applied. The cells were maintained by cell bleeds at density 0.9–1.3 × 10⁸ cells/mL in growing state and at high viability for more than 2 weeks. Finally, with the present settings, maximal cell densities of 2.14 × 10⁸ cells/mL, achieved for the first time in a wave‐induced bioreactor, and 1.32 × 10⁸ cells/mL were reached using TFF and ATF systems, respectively. Using TFF, the cell density was limited by the membrane capacity for the encountered high viscosity and by the pCO₂ level. Using ATF, the cell density was limited by the vacuum capacity failing to pull the highly viscous fluid. Thus, the TFF system allowed reaching higher cell densities. The TFF inlet pressure was highly correlated to the viscosity leading to the development of a model of this pressure, which is a useful tool for hollow fiber design of TFF and ATF. At very high cell density, the viscosity introduced physical limitations. This led us to recommend cell densities under 1.46 × 10⁸ cell/mL based on the analysis of the theoretical distance between the cells for the present cell line. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:754–767, 2013