Varying Cell Densities Research Articles

Transport of self-propelling bacteria in micro-channel flow

Understanding the collective motion of self-propelling organisms in confined geometries, such as that of narrow channels, is of great theoretical and practical importance. By means of numerical simulations we study the motion of model bacteria in 2D channels under different flow conditions: fluid at rest, steady and unsteady flow. We find aggregation of bacteria near channel walls and, in the presence of external flow, also upstream swimming, which turns out to be a very robust result. Detailed analysis of bacterial velocity and orientation fields allows us to quantify the phenomenon by varying cell density, channel width and fluid velocity. The tumbling mechanism turns out to have strong influence on velocity profiles and particle flow, resulting in a net upstream flow in the case of non-tumbling organisms. Finally we demonstrate that upstream flow can be enhanced by a suitable choice of an unsteady flow pattern.

Analyzing the metabolic stress response of recombinant Escherichia coli cultures expressing human interferon-beta in high cell density fed batch cultures using time course transcriptomic data

Fed batch cultures expressing recombinant interferon beta under the T7 promoter were run with different exponential feeding rates of a complex substrate and induced at varying cell densities. Post-induction profiles of the specific product formation rates showed a strong dependence on the specific growth rate with the maximum product yield obtained at 0.2 h(-1). A study of the relative transcriptomic profiles as a function of pre-induction μ was therefore done to provide insight into the role of cellular physiology in enhancing recombinant protein expression. Hierarchical clustering analysis of the significantly regulated genes allowed us to identify biologically important groups of genes which fall under specific master regulators. The groups were: rpoH, ArcB, CreB, Lrp, RelA, Fis and Hfq. The response of these regulators, which exert a feedback control on the growth and product formation rates correlated well with the expression levels obtained. Thus at the optimum pre-induction μ, the alternative sigma factors and ribosomal machinery genes did not get depressed till the 6th hour post-induction unlike at other specific growth rates, demonstrating a critical role for the genes in sustaining recombinant protein expression.

Inferring computational function of neuronal networks from multi-electrode array recordings: an evolutionary approach

The availability of microelectrode array systems (MEA’s) has increased dramatically in recent years. Along with this increase in availability, these systems have also grown in capability. Modern systems stimulate and record in-vitro neuronal networks on an increasing number of channels. Closed-loop capabilities further expand the functionality of these systems. Future miniaturization of these systems and commensurate increased resolution promises a continued rise in the volume of data being produced in this domain. It is difficult to determine if analysis tools are keeping pace with data generation. As of a few years ago Brown et al. saw a distinct need for investment in this vein[1]. It seems likely that data acquisition will continue to outstrip analysis just as it has in other bioinformatics domains. In 2006 Waganaar et al made available an extensive data set or recorded microelectrode array activity [2]. This data set contained both spontaneous and stimulated activity recorded regularly, from 58 unique cultures, of varying cell density, over a period of five weeks. While this study is referenced by hundreds of presenters and researchers, only a few groups have published analyses of this data. Patnaik et al stand out in that very small cohort as they sought to infer network structure from this data set based on the last 5 days of recordings from 6 cultures[3]. It is likely that these cultures were more static at this stage and were no longer developing connections at the same rate one may have observed in earlier time points. Genetic algorithms (GA’s) and other evolutionary computing techniques have a proven track record in temporal knowledge discovery, network analysis, and machine learning[4][5]. In this work we demonstrate the feasibility of using a GA to tease out temporal and spatial relationships in the neuronal networks. Our analysis yields a functional and explanatory model of signal propagation on MEA’s and the development of neuronal networks in-vitro. As shown in figure ​figure11 we seek to incorporate a broad range of MEA activity. The descriptive nature of the output from the evolutionary system can be tuned to include domain specific inferences. These models can present more actionable information for network design than their purely statistical counterparts. Figure 1 Generalized system view

Cell-density-dependent changes in mitochondrial membrane potential and reactive oxygen species production in human skin cells post sunlight exposure

Solar ultraviolet radiation (UVR) is the principal etiological factor in skin carcinogenesis. In vivo and in vitro studies have demonstrated previously that oxidative DNA damage, mitochondrial mass and mitochondrial membrane potential (MMP) changes are associated with skin cell response to UVR stress. Spontaneously immortalized human skin keratinocytes were irradiated with increasing sub-lethal doses of simulated sunlight irradiation (SSI) using a Q-Sun solar simulator. The effects of SSI on reactive oxygen species (ROS) formation, mitochondrial mass and MMP were then determined. SSI induced mitochondrial mass increase post low SSI (0.25-2.5 J/cm²), whereas higher SSI doses (5.0 and 7.5 J/cm²) decreased mitochondrial mass. Mitochondrial mass increased with time post 5.0 J/cm² irradiation and all changes in mass were independent of cell density status. Changes in ROS and MMP were cell density dependent. Additionally, an inverted dose-dependent decrease in ROS formation was observed 3 h post SSI with the lower SSI dose (0.25 J/cm²). Observations from the present study suggest that changes in the cell's microenvironment (modeled through varying cell density) influence changes in MMP and ROS detoxifying responses in sun-exposed skin cells.

Na+-dependent HCO3− Import by the slc4a10 Gene Product Involves Cl− Export

The slc4a10 gene encodes an electroneutral Na(+)-dependent HCO(3)(-) importer for which the precise mode of action remains unsettled. To resolve this issue, intracellular pH (pH(i)) recordings were performed upon acidification in the presence of CO(2)/HCO(3)(-) by 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) fluorometry of stably slc4a10-transfected NIH-3T3 fibroblasts. slc4a10 expression induced a significant Na(+)-dependent pH(i) recovery, which was accompanied by an increase in the intracellular Na(+) concentration evaluated by use of the Na(+)-sensitive fluorophore CoroNa Green. The estimated Na(+):HCO(3)(-) stoichiometry was 1:2. Cl(-) is most likely the counterion maintaining electroneutrality because (i) Na(+)-dependent pH(i) recovery was eliminated in Cl(-)-depleted cells; (ii) acute extracellular Cl(-) removal led to a larger alkalization in slc4a10-transfected cells than in control cells; and (iii) the 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS)-sensitive and Na(+)- and HCO(3)(-)-dependent (36)Cl(-)-efflux during pH(i) recovery was significantly greater in acidified slc4a10-transfected cells than in control cells. Charged amino acids specific to slc4a gene family members that transport Na(+) and are expected to move more HCO(3)(-) molecules/turnover were targeted by site-directed mutagenesis. Na(+)-dependent pH(i) recovery was reduced in each of the single amino acid mutated cell lines (E890A, E892A, H976L, and H980G) compared with wild type slc4a10-transfected cells and completely eliminated in quadruple mutant cells. In conclusion, the data suggest that slc4a10 expressed in mammalian cells encodes a Na(+)-dependent Cl(-)/HCO(3)(-) exchanger in which four specific charged amino acids seem necessary for ion transport.

Cell contraction forces in scaffolds with varying pore size and cell density

The contractile behavior of cells is relevant in understanding wound healing and scar formation. In tissue engineering, inhibition of the cell contractile response is critical for the regeneration of physiologically normal tissue rather than scar tissue. Previous studies have measured the contractile response of cells in a variety of conditions (e.g. on two-dimensional solid substrates, on free-floating tissue engineering scaffolds and on scaffolds under some constraint in a cell force monitor). Tissue engineering scaffolds behave mechanically like open-cell elastomeric foams: between strains of about 10 and 90%, cells progressively buckle struts in the scaffold. The contractile force required for an individual cell to buckle a strut within a scaffold has been estimated based on the strut dimensions (radius, r, and length, l) and the strut modulus, Es. Since the buckling force varies, according to Euler's law, with r4/l2, and the relative density of the scaffold varies as (r/l)2, the cell contractile force associated with strut buckling is expected to vary with the square of the pore size for scaffolds of constant relative density. As the cell density increases, the force per cell to achieve a given strain in the scaffold is expected to decrease. Here we model the contractile response of fibroblasts by analyzing the response of a single tetrakaidecahedron to forces applied to individual struts (simulating cell contractile forces) using finite element analysis. We model tetrakaidecahedra of different strut lengths, corresponding to different scaffold pore sizes, and of varying numbers of loaded struts, corresponding to varying cell densities. We compare our numerical model with the results of free-floating contraction experiments of normal human dermal fibroblasts (NHDF) in collagen-GAG scaffolds of varying pore size and with varying cell densities.

Cortical Neural Precursors Inhibit Their Own Differentiation via N-Cadherin Maintenance of β-Catenin Signaling

Little is known about the architecture of cellular microenvironments that support stem and precursor cells during tissue development. Although adult stem cell niches are organized by specialized supporting cells, in the developing cerebral cortex, neural stem/precursor cells reside in a neurogenic niche lacking distinct supporting cells. Here, we find that neural precursors themselves comprise the niche and regulate their own development. Precursor-precursor contact regulates beta-catenin signaling and cell fate. In vivo knockdown of N-cadherin reduces beta-catenin signaling, migration from the niche, and neuronal differentiation in vivo. N-cadherin engagement activates beta-catenin signaling via Akt, suggesting a mechanism through which cells in tissues can regulate their development. These results suggest that neural precursor cell interactions can generate a self-supportive niche to regulate their own number.

Estimation of average acoustic concentration of Chinese hamster ovary cells using ultrasonic backscatter.

Average acoustic concentration (AAC) is one important parameter of quantitative ultrasonic assessment of soft tissues. The objective of this study is to examine the AAC of Chinese hamster ovary (CHO) cells at varied cell densities using ultrasonic backscatter. The CHO cells were evenly distributed in 2% agar and the densities ranged from 500,000 to 54,000,000 cells/mL. Measurements of backscattered ultrasound were obtained over the frequency range of 17 to 80 MHz. Estimates of Average Scatterer Diameters and AAC were made by comparing the backscattered power spectra with three scattering models: Fluid‐filled Sphere model (FFSM), Spherical Gaussian model (SGM), and Concentric Sphere Model (CSM). Over the frequency range of 28 to 50 MHz, the estimated ASDs were 20.0±0 μm with FFSM, 22.2±6.0 μm with SGM, and 20.1±6.1 μm with CSM. In the cell density range of 500,000 to 10,000,000 cells/mL, the estimated AACs increased almost linearly from 10 to 34 dB/mm3 using FFSM, from 16 to 39 dB/mm3 using SGM, and from 8...

Ex vivo Expansion and Differentiation of Mesenchymal Stem Cells from Goat Bone Marrow

Objective(s) Mesenchymal stem cells (MSCs) from large animals as goat which is genetically more closely related to human have rarely been gained attentions. The present study tried to isolate and characterize MSCs from goat bone marrow. Materials and Methods Fibroblastic cells appeared in goat marrow cell culture were expanded through several subcultures. Passaged-3 cells were then differentiated among the osteogenic, adipogenic and chondrogenic cell lineages to determine their MSC nature. Differentiations were determined by RT-PCR analysis of related gene expression. To identify the best culture conditions for propagation, passage-3 cells were plated either at varying cell densities or different fetal bovine serum (FBS) concentrations for a week, at the end of which the cultures were statistically compared with respect to the cell proliferation. In this study, we also determined goat MSC population doubling time (PDT) as the index of their in vitro expansion rate. Results Passage-3 fibroblastic cells tended to differentiate into skeletal cell lineages. This was evident in both specific staining as well as the specific gene expression profile. Moreover, there appeared to be more expansion when the cultures were initiated at 100 cells/cm[1] in a medium supplemented with 15% FBS. A relatively short PDT (24.94±2.67 hr) was a reflection of the goat MSC rapid rate of expansion. Conclusion Taken together, fibroblastic cells developed at goat marrow cell culture are able to differentiate into skeletal cell lineages. They undergo extensive proliferation when being plated at low cell density in 15% FBS concentration.

Impact of cell density on microbially induced stable isotope fractionation

Quantification of microbial contaminant biodegradation based on stable isotope fractionation analysis (SIFA) relies on known, invariable isotope fractionation factors. The microbially induced isotope fractionation is caused by the preferential cleavage of bonds containing light rather than heavy isotopes. However, a number of non-isotopically sensitive steps preceding the isotopically sensitive bond cleavage may affect the reaction kinetics of a degradation process and reduce the observed (i.e., the macroscopically detectable) isotope fractionation. This introduces uncertainty to the use of isotope fractionation for the quantification of microbial degradation processes. Here, we report on the influence of bacterial cell density on observed stable isotope fractionation. Batch biodegradation experiments were performed under non-growth conditions to quantify the toluene hydrogen isotope fractionation by exposing Pseudomonas putida mt-2(pWWO) at varying cell densities to different concentrations of toluene. Observed isotope fractionation depended significantly on the cell density. When the cell density rose from 5 x 10(5) to 5 x 10(8)cells/mL, the observed isotope fractionation declined by 70% and went along with a 55% decrease of the degradation rates of individual cells. Theoretical estimates showed that uptake-driven diffusion to individual cells depended on cell density via the overlap of the cells' diffusion-controlled boundary layers. Our data suggest that biomass effects on SIFA have to be considered even in well-mixed systems such as the cell suspensions used in this study.

Large quantity cryopreservation of bovine testicular cells and its effect on enrichment of type A spermatogonia

Cryopreservation has become an integral component of any cell transplantation technique helping to overcome the issues associated with known spatial and temporal barriers between donor and recipient. The aim of this study was to develop a protocol for large quantity cryopreservation of bovine testicular germ cells. The impact of 3 different packaging methods (5 ml semen straw, 20 ml freezing bag and 1.5 ml cryovial) and varying cell densities (3 × 10 6, 9 × 10 6, or 18 × 10 6 cells/ml) on the survival of testis germ cells was examined. Cells processed in 5 ml semen straws had a significantly higher viability (70.7 ± 1.2%, P < 0.05) compared to those cells in 20 ml freezing bags (46.7 ± 0.1%) or 1.5 ml cryovials (46.3 ± 2.2%). For 5 ml straws, a 20 min cooling prior to cryopreservation resulted in a higher post thaw viability (73.2 ± 0.6%) than a 10 min cooling (56.0 ± 2.2%), while the density of the cell suspension did not impact on post thaw viability. Thus cryopreservation of testicular germ cells in 5 ml straws at a density between 3 × 10 6 and 18 × 10 6 cells/ml in liquid nitrogen vapour for 20 min cooling appears to be a simple and practical way to preserve cells. Subsequent testing of frozen/thawed cells exhibited viable cultures and retained the ability to proliferate. The freezing protocol does not preferentially preserve type A spermatogonia. However, the cell surface properties of somatic cells appear to be affected by the freezing procedure and therefore the frozen/thawed cells are less suitable for enriching type A spermatogonia by differential plating.

Role for sagA and siaA in Quorum Sensing and Iron Regulation in Streptococcus pyogenes

Streptococcus pyogenes is a ubiquitous and versatile pathogen that causes a variety of infections with a wide range of severity. The versatility of this organism is due in part to its capacity to regulate virulence gene expression in response to the many environments that it encounters during an infection. We analyzed the expression of two potential virulence factors, sagA and siaA (also referred to as pel and htsA, respectively), in response to conditions of varying cell densities and iron concentrations. The sagA gene was up-regulated in conditioned medium from a wild-type strain but not from sagA-deficient mutants, and the gene was also up-regulated in the presence of streptolysin S (SLS), the gene product of sagA, thus indicating that this gene or its product is involved in density-dependent regulation of S. pyogenes. By comparison, siaA responded in a manner consistent with a role in iron acquisition since it was up-regulated under iron-restricted conditions. Although siaA expression was also up-regulated in the presence of SLS and in conditioned media from both wild-type and sagA-deficient mutants, this up-regulation was not growth phase dependent. We conclude that sagA encodes a quorum-sensing signaling molecule, likely SLS, and further support the notion that siaA is likely involved in iron acquisition.

Control of the Bioluminescence Starting Time by Inoculated Cell Density

In this study, we attempted to control the timing of light-emission from bioluminescent bacteria, by changed cell numbers inoculated into medium. Luminous bacteria express bioluminescence when the number of cells reached a threshold. Inoculated cell density had an effect on the time of bioluminescence starting. Samples were prepared by varying cell density of inoculation. In the results, all the vials showed different luminescence profiles in the order of inoculated cell population.

Interferon γ–Inducible Protein 10 Selectively Inhibits Proliferation and Induces Apoptosis in Endothelial Cells

Interferon gamma-inducible protein 10 (IP-10) has antitumor effects in various murine models. The IP-10 receptor has two distinct splice variants, CXCR3A and CXCR3B, that have paradoxical effects after ligand-receptor interaction. To characterize the putative antiangiogenic effects of IP-10, we measured proliferation rates and apoptosis in human umbilical vein endothelial cells (HUVECs), fibroblasts, and A375 melanoma or WIDR adenocarcinoma cell lines after exposure to the recombinant protein. CXCR3A (activating) and CXCR3B (inhibitory/proapoptotic) messenger RNA (mRNA) expression levels in fibroblasts, 2 human tumor cell lines, T lymphocytes, and HUVECs of varying cell densities were characterized. IP-10 resulted in dose-dependent and selective inhibition of proliferation and countered the proliferative effects of vascular endothelial growth factor in HUVECs but did not affect fibroblasts or 2 human tumor cell lines. In addition, IP-10 resulted in potent and selective induction of apoptosis in HUVECS but had no effect on fibroblasts or A375 melanoma. Confluent HUVECs had a predominance of mRNA for the CXCR3B splice variant by reverse transcriptase-polymerase chain reaction, and the ratio of CXCR3B to CXCR3A mRNA was >40 in HUVECs, compared with </=10 in the other cell types. Moreover, CXCR3B mRNA levels were significantly higher in proliferating compared with confluent HUVECs. In vivo, systemic IP-10 administration resulted in slower A375 xenograft growth rates compared with control-treated animals, and immunohistochemical staining showed decreased microvessel density in xenografts of IP-10-treated mice. IP-10 has antiangiogenic properties and selective effects on endothelial tissue that may be secondary to higher levels of the CXCR3B inhibitory/proapoptotic receptor in that cell type, particularly in its actively proliferating state.

Regulation of the poly(ADP-ribose) polymerase-1 gene expression by the transcription factors Sp1 and Sp3 is under the influence of cell density in primary cultured cells

PARP-1 [poly(ADP-ribose) polymerase-1) is a nuclear enzyme that is involved in several cellular functions, including DNA repair, DNA transcription, carcinogenesis and apoptosis. The activity directed by the PARP-1 gene promoter is mainly dictated through its recognition by the transcription factors Sp1 and Sp3 (where Sp is specificity protein). In the present study, we investigated whether (i) both PARP-1 expression and PARP-1 enzymatic activity are under the influence of cell density in primary cultured cells, and (ii) whether its pattern of expression is co-ordinated with that of Sp1/Sp3 at varying cell densities and upon cell passages. All types of cultured cells expressed PARP-1 in Western blot when grown to sub-confluence. However, a dramatic reduction was observed at post-confluence. Similarly, high levels of Sp1/Sp3 were observed by both Western blot and EMSAs (electrophoretic mobility-shift assays) in sub-confluent,but not post-confluent, cells. Consistent with these results, the promoter of the rPARP-1 (rat PARP-1) gene directed high levels of activity in sub-confluent, but not confluent, cells upon transfection of various CAT (chloramphenicol acetyltransferase)-rPARP-1 promoter constructs into cultured cells. The positive regulatory influence of Sp1 was not solely exerted on the rPARP-1 promoter constructs, as inhibition of endogenous Sp1 expression in HDKs(human dermal keratinocytes) through the transfection of Sp1 RNAi (RNA interference) considerably reduced endogenous hPARP-1 (human PARP-1) expression as well. The reduction in PARP-1 protein expression as cells reached confluence also translated into a corresponding reduction in PARP-1 activity. In addition, expression of both Sp1/Sp3, as well as that of PARP-1,was dramatically reduced as cells were passaged in culture and progressed towards irreversible terminal differentiation. PARP-1 gene expression therefore appears to be co-ordinated with that of Sp1 and Sp3 in primary cultured cells, suggesting that PARP-1 may play some important functions during the proliferative burst that characterizes wound healing.

An application of CFD to improve warm-up performance of the 3-way auto-catalyst by high surface area and low thermal mass

Mathematical modelling of three-way catalytic converter (3WCC) operation is used increasingly in the optimisation of automobile converter systems. But almost all of previous computational models were based on an one-channel approach with the reaction kinetics computations, which is useful and efficient in predicting real-world performance of the catalyst. However, as long as flow maldistribution is not accounted for in the models, simulation results will not be reliable. In this work, 2-dimensional performance prediction of catalyst has been performed by using turbulent flow simulation coupled with reaction mechanism for surface chemistry and the results were compared with experimental data and one channel simulation in the literature. The computational results from this study show the better prediction accuracy in terms of CO, HC and NO conversion efficiencies compared to those of the 1-D adiabatic model. Varying cell density and hot spot moving pattern within the monolith during warm-up period are also considered. The results indicate that the higher cell density giving much larger geometric surface area shows the faster movement of thermal front of hot spot due to the higher convective heat transfer rate.

Cell Density Regulated Expression of Transcription Factor Sp1 in Corneal Stromal Cultures

Sp1, a ubiquitously expressed transcription factor, has been implicated to have a role in cell differentiation and cell proliferation. In keratoconus, a corneal disease characterized by thinning and scarring of the central cornea, Sp1 is found up-regulated. In the present study, we examined the expression of Sp1 in stromal cells cultured from normal human and keratoconus-afflicted corneas and evaluated the influence of varying cell densities. Immunohistochemical staining, Western blotting and electrophoretic mobility shift assays indicated that in both normal human and keratoconus cultures, Sp1 protein levels and binding activities increased with the density of cells. The basal level of Sp1 in keratoconus cultures was higher than that in normals. These results demonstrate a marked density mediated up-regulation of Sp1 in corneal stromal cells, suggesting that the Sp1 expression may be regulated by differentiation states of the cells in the cornea. In addition, cells from keratoconus corneas in vitro appear to carry and retain the Sp1 abnormality as in vivo. The Sp1 defect may be an inborn error in keratoconus.

An analytical approach to the measurement of equilibrium binding constants: application to EGF binding to EGF receptors in intact cells measured by flow cytometry.

In ligand binding studies, ligand depletion often limits the accuracy of the results obtained. This problem is approached by employing the simple observation that as the concentration of receptor in the assay is reduced, ligand depletion is also reduced. Measuring apparent K(D)'s of a ligand at multiple concentrations of receptor with extrapolation to infinitely low receptor concentration takes ligand depletion into account and, depending on the binding model employed, yields a K(D) within the defined limits of accuracy. We apply this analysis to the binding of epidermal growth factor (EGF) to the EGF receptor expressed in intact 32D cells, using a homogeneous fluorescein-labeled preparation of EGF and measuring binding by flow cytometry. Binding isotherms were carried out at varying cell densities with each isotherm fit to the generally applied model with two independent binding sites. Examination of the variation in the K(D)'s versus cell density yields a high-affinity site that accounts for 18% of the sites and a lower affinity site that accounts for the remainder. However, further examination of these data suggests that while consistent with each individual isotherm, the simple model of two independent binding sites that is generally applied to EGF binding to the EGF receptor is inconsistent with the changes in the apparent K(D)'s seen across varying cell densities.

False Positivity in a Cyto-ELISA for Anti-Endothelial Cell Antibodies Caused by Heterophile Antibodies to Bovine Serum Proteins

ELISAs with fixed endothelial cells or cell lines are widely used screening tests for anti-endothelial cell antibodies (AECAs), but spurious increases occur. We examined interferences by heteroantibodies and means to eliminate them. AECAs were measured by ELISA on fixed layers of the human endothelial cell line, EA.hy 926, in a panel of 60 patient serum samples diluted in bovine serum albumin. Heteroantibodies against fetal calf serum (FCS) proteins were demonstrated and characterized in an ELISA-the interference assay-that used FCS-coated plates and Tween 20-containing buffer as blocking agent and sample diluent, as well as by immunoblotting. In 12 of 60 patient serum samples, spurious increases of AECA titers were produced by endogenous antibodies reacting with FCS proteins from culture medium that were coated onto the solid-phase at the time of cell plating. This mechanism of interference was supported experimentally by exposing extracellular matrix, varying cell density, and incubating wells with FCS alone. The heterophile antibodies were mainly IgG and IgA, and in inhibition experiments, they recognized serum proteins from goat, sheep, and horse. Washing cells free of FCS before plating, or adding FCS (100 mL/L) to the patient sample diluent eliminated spurious signals from all 30 tested sera, but the latter method had practical advantages. Antibodies against animal serum proteins are a frequent cause of erroneous results in cyto-ELISAs. The interference can be eliminated by simple antibody absorption in FCS-containing dilution buffer.

Modeling Pichia pastoris growth on methanol and optimizing the production of a recombinant protein, the heavy-chain fragment C of botulinum neurotoxin, serotype A.

An unstructured growth model for the recombinant methylotrophic yeast P. pastoris Mut(+) expressing the heavy-chain fragment C of botulinum neurotoxin serotype A [BoNT/A(H(c))], was successfully established in quasi-steady state fed-batch fermentations with varying cell densities. The model describes the relationships between specific growth rate and methanol concentration, and the relationships between specific methanol and ammonium consumption rates and specific growth rate under methanol-limited growth conditions. The maximum specific growth rate (mu) determined from the model was 0.08 h(-1) at a methanol concentration of 3.65 g/L, while the actual maximum mu was 0.0709 h(-1). The maximum specific methanol consumption rate was 0.0682 g/g WCW/h. From the model, growth can be defined as either methanol-limited or methanol-inhibited and is delineated at a methanol concentration of 3.65 g/L. Under inhibited conditions, the observed biomass yield (Y(X/MeOH)) was lower and the maintenance coefficient (m(MeOH)) was higher than compared to limited methanol conditions. The Y(X/MeOH) decreased and m(MeOH) increased with increasing methanol concentration under methanol-inhibited conditions. BoNT/A(H(c)) content in cells (alpha) under inhibited growth was lower than that under limited growth, and decreased with increasing methanol concentration. A maximum alpha of 1.72 mg/g WCW was achieved at a mu of 0.0267 h(-1) and induction time of 12 h.