Biomedical subjects

James M Piret

Publications and source records attributed to James M Piret.

12 recordsLinked to original sources

PubMed2007

Decreased pCO(2) accumulation by eliminating bicarbonate addition to high cell-density cultures.

High-density perfusion cultivation of mammalian cells can result in elevated bioreactor CO(2) partial pressure (pCO(2)), a condition that can negatively influence growth, metabolism, productivity, and protein glycosylation. For BHK cells in a perfusion culture at 20 x 10(6) cells/mL, the bioreactor pCO(2) exceeded 225 mm Hg with approximate contributions of 25% from cellular respiration, 35% from medium NaHCO(3), and 40% from NaHCO(3) added for pH control. Recognizing the limitations to the practicality of gas sparging for CO(2) removal in perfusion systems, a strategy based on CO(2) reduction at the source was investigated. The NaHCO(3) in the medium was replaced with a MOPS-Histidine buffer, while Na(2)CO(3) replaced NaHCO(3) for pH control. These changes resulted in 63-70% pCO(2) reductions in multiple 15 L perfusion bioreactors, and were reproducible at the manufacturing-scale. Bioreactor pCO(2) values after these modifications were in the 68-85 mm Hg range, pCO(2) reductions consistent with those theoretically expected. Low bioreactor pCO(2) was accompanied by both 68-123% increased growth rates and 58-92% increased specific productivity. Bioreactor pCO(2) reduction and the resulting positive implications for cell growth and productivity were brought about by process changes that were readily implemented and robust. This philosophy of pCO(2) reduction at the source through medium and base modification should be readily applicable to large-scale fed-batch cultivation of mammalian cells.

Animals↗

PubMed2006

Meta-analysis of differentiating mouse embryonic stem cell gene expression kinetics reveals early change of a small gene set.

Stem cell differentiation involves critical changes in gene expression. Identification of these should provide endpoints useful for optimizing stem cell propagation as well as potential clues about mechanisms governing stem cell maintenance. Here we describe the results of a new meta-analysis methodology applied to multiple gene expression datasets from three mouse embryonic stem cell (ESC) lines obtained at specific time points during the course of their differentiation into various lineages. We developed methods to identify genes with expression changes that correlated with the altered frequency of functionally defined, undifferentiated ESC in culture. In each dataset, we computed a novel statistical confidence measure for every gene which captured the certainty that a particular gene exhibited an expression pattern of interest within that dataset. This permitted a joint analysis of the datasets, despite the different experimental designs. Using a ranking scheme that favored genes exhibiting patterns of interest, we focused on the top 88 genes whose expression was consistently changed when ESC were induced to differentiate. Seven of these (103728_at, 8430410A17Rik, Klf2, Nr0b1, Sox2, Tcl1, and Zfp42) showed a rapid decrease in expression concurrent with a decrease in frequency of undifferentiated cells and remained predictive when evaluated in additional maintenance and differentiating protocols. Through a novel meta-analysis, this study identifies a small set of genes whose expression is useful for identifying changes in stem cell frequencies in cultures of mouse ESC. The methods and findings have broader applicability to understanding the regulation of self-renewal of other stem cell types.

Adaptation, Physiological↗

PubMed2005

Accelerating perfusion process optimization by scanning non-steady-state responses.

Perfusion processes provide consistent culture conditions, high productivity and low product residence times. However, process development can be slow due to the 1 week or more required to reach each steady state. The objective of this work was to accelerate process development in perfusion cultures by scanning non-steady-state transient responses to qualitatively predict steady-state performance. The method was tested using a shift in temperature every 3 days, scanned down by steps of 2 degrees C from 37 degrees C to 31 degrees C, then scanned up to 37 degrees C. Higher t-PA concentrations were predicted at lower temperatures, confirmed by subsequent pseudo-steady-state results. In most cases, transient values on the 3rd day were in close concordance with pseudo-steady-state values. To further accelerate process development, transient scanning was applied to small-scale, non-instrumented cultures. Similar results were obtained, although quantitative t-PA values were 15-30 times lower than in high cell density perfusion cultures. The method was further explored by investigating 1 day transient shifts in temperature where more variability was observed, suggesting that the cells were still adapting to the new environment. Nonetheless, the overall response again qualitatively predicted the pseudo-steady-state temperature response. Use of transient scanning in conjunction with pseudo-steady-state verification and refinement of optimal results could reduce process development time to a third or less of comparable steady-state-based optimization.

Animals↗

PubMed2005

Optical analysis of perfusion bioreactor cell concentration in an acoustic separator.

Automated monitoring of cell concentration in perfusion bioprocesses facilitates the maintenance of constant cell specific perfusion rates. However, most on-line measuring devices are relatively complex and foul as the culture progresses. A simple external optical sensor was developed using the transparent glass walls of acoustic separators for automated optical analysis of their contents. For each measurement, the separator was filled by an automated pumping system with triplicate representative bioreactor samples that were optically analyzed and the device returned to perfusion operation within approximately 1 or 2 min. Chinese hamster ovary cell concentrations, ranging from 5 x 10(5) to 2 x 10(7) cells/mL, were highly correlated (R(2) = 0.99) with the 90 degrees scattered light response. Since the device was operated externally, it did not complicate bioreactor sterilization or cleaning. Viability was not optically analyzed, but this information was not required between manual samples of a properly operated perfusion process. Using single-point recalibration based on routine off-line samples, this external optical system remained effective during a 4-month perfusion run, thus providing a non-invasive and easily maintained on-line cell concentration monitoring system to improve the control of perfusion bioreactors.

Acoustics↗

PubMed2005

Characterization and optimization of acoustic filter performance by experimental design methodology.

Acoustic cell filters operate at high separation efficiencies with minimal fouling and have provided a practical alternative for up to 200 L/d perfusion cultures. However, the operation of cell retention systems depends on several settings that should be adjusted depending on the cell concentration and perfusion rate. The impact of operating variables on the separation efficiency performance of a 10-L acoustic separator was characterized using a factorial design of experiments. For the recirculation mode of separator operation, bioreactor cell concentration, perfusion rate, power input, stop time and recirculation ratio were studied using a fractional factorial 2(5-1) design, augmented with axial and center point runs. One complete replicate of the experiment was carried out, consisting of 32 more runs, at 8 runs per day. Separation efficiency was the primary response and it was fitted by a second-order model using restricted maximum likelihood estimation. By backward elimination, the model equation for both experiments was reduced to 14 significant terms. The response surface model for the separation efficiency was tested using additional independent data to check the accuracy of its predictions, to explore robust operation ranges and to optimize separator performance. A recirculation ratio of 1.5 and a stop time of 2 s improved the separator performance over a wide range of separator operation. At power input of 5 W the broad range of robust high SE performance (95% or higher) was raised to over 8 L/d. The reproducible model testing results over a total period of 3 months illustrate both the stable separator performance and the applicability of the model developed to long-term perfusion cultures.

Acoustics↗

PubMed2005

Correlation of murine embryonic stem cell gene expression profiles with functional measures of pluripotency.

Global gene expression profiling was performed on murine embryonic stem cells (ESCs) induced to differentiate by removal of leukemia inhibitory factor (LIF) to identify genes whose change in expression correlates with loss of pluripotency. To identify appropriate time points for the gene expression analysis, the dynamics of loss of pluripotency were investigated using three functional assays: chimeric mouse formation, embryoid body generation, and colony-forming ability. A rapid loss of pluripotency was detected within 24 hours, with very low residual activity in all assays by 72 hours. Gene expression profiles of undifferentiated ESCs and ESCs cultured for 18 and 72 hours in the absence of LIF were determined using the Affymetrix GeneChip U74v2. In total, 473 genes were identified as significantly differentially expressed, with approximately one third having unknown biological function. Among the 275 genes whose expression decreased with ESC differentiation were several factors previously identified as important for, or markers of, ESC pluripotency, including Stat3, Rex1, Sox2, Gbx2, and Bmp4. A significant number of the decreased genes also overlap with previously published mouse and human ESC data. Furthermore, several membrane proteins were among the 48 decreased genes correlating most closely with the functional assays, including the stem cell factor receptor c-Kit. Through identification of genes whose expression closely follows functional properties of ESCs during early differentiation, this study lays the foundation for further elucidating the molecular mechanisms regulating the maintenance of ESC pluripotency and facilitates the identification of more reliable molecular markers of the undifferentiated state.

Animals↗

PubMed2004

Empirical models of the proliferative response of cytokine-dependent hematopoietic cell lines.

There is an expanding need for predictive mathematical models to accelerate the optimization of cell therapy culture processes. Here we demonstrate the ability of simple mathematical models to describe quantitatively the cytokine growth-rate dependence of two human hematopoietic cell lines, TF-1 and MO7e. These cells are immortal but depend on either interleukin-3 (IL-3) or granulocyte-macrophage colony stimulating factor (GM-CSF) for their continued survival and maximal proliferation. They are also responsive to interleukin-6 (IL-6) and exhibit saturation kinetics when these cytokines are limiting. A Monod-type relationship consistently failed to fit measured cytokine dose-proliferation response curves while a Hill-type relationship showed a good fit. Cytokine interactions were first modeled by modifying the Hill-function to include an interaction parameter, gamma. This model did not indicate either synergistic or even additive effects between IL-3 and GM-CSF. Based on the reported competition between IL-3 and GM-CSF for their common receptor (beta(c)) subunit, a competitive model was also developed. This model had no new parameters beyond those obtained from single cytokine cultures and provided improved prediction of the growth rates for both cell lines exposed to combinations of IL-3 and GM-CSF over a wide range of concentrations. As expected, the competitive model failed to fit the data for IL-6 in combination with either IL-3 or GM-CSF, since IL-6 signaling does not involve the beta(c) chain of the IL-3/GM-CSF receptors. Interestingly, the cell-specific rates of GM-CSF uptake and cell proliferation were found to be uncoupled processes. Taken together, these results illustrate the utility of appropriately designed empirical models to describe the proliferative responses of hematopoietic cells to cytokine stimulation.

Cell Culture Techniques↗

PubMed2002

Scale-up and optimization of an acoustic filter for 200 L/day perfusion of a CHO cell culture.

Acoustic cell retention devices have provided a practical alternative for up to 50 L/day perfusion cultures but further scale-up has been limited. A novel temperature-controlled and larger-scale acoustic separator was evaluated at up to 400 L/day for a 10(7) CHO cell/mL perfusion culture using a 100-L bioreactor that produced up to 34 g/day recombinant protein. The increased active volume of this scaled-up separator was divided into four parallel compartments for improved fluid dynamics. Operational settings of the acoustic separator were optimized and the limits of robust operations explored. The performance was not influenced over wide ranges of duty cycle stop and run times. The maximum performance of 96% separation efficiency at 200 L/day was obtained by setting the separator temperature to 35.1 degrees C, the recirculation rate to three times the harvest rate, and the power to 90 W. While there was no detectable effect on culture viability, viable cells were selectively retained, especially at 50 L/day, where there was a 5-fold higher nonviable washout efficiency. Overall, the new temperature-controlled and scaled-up separator design performed reliably in a way similar to smaller-scale acoustic separators. These results provide strong support for the feasibility of much greater scale-up of acoustic separations.

Acoustics↗

PubMed2002

Common and distinct features of cytokine effects on hematopoietic stem and progenitor cells revealed by dose-response surface analysis.

Recent studies have identified thrombopoietin (TPO), flt-3 ligand (FL), Steel factor (SF), and interleukin-11 (IL-11) as cytokines able to stimulate amplification of the most primitive murine hematopoietic cells in vitro. However, dose-response and interaction parameters that predict how to optimize mixtures of these cytokines have not been previously defined. To obtain this information, Sca-1(+)lin(-) and c-kit(+)Sca-1(+)lin(-) adult mouse bone marrow cells were cultured for 10 and 14 days, respectively, in serum-free medium with varying concentrations of these cytokines. Quantitative assays were performed to determine the influences of the cytokine combinations tested on changes in long-term repopulating hematopoietic stem cells (HSCs), in vitro colony-forming cells (CFCs), and total cell numbers. A two-level factorial design was first used to screen the effects of TPO, SF, FL, and IL-11 as well as two different incubation temperatures. IL-11 and SF were found to be the most significant stimulators of murine HSC expansion. More detailed analyses of the effects on c-kit(+)Sca-1(+)lin(-) cells of IL-11, SF, and FL concentrations and their interactions using response surface methodology showed IL-11 to have a maximal stimulatory effect on HSC expansion at 20 ng/mL with higher concentrations being inhibitory. In contrast, not even high concentration saturation of the effects of either SF or FL was observed as the stimulatory effect of both SF and FL increased beyond 300 ng/mL. A negative interaction between SF and FL on HSCs was discovered. Interestingly, a generally similar pattern of cytokine effects was found to influence the 14-day output of CFCs and total cells from the same c-kit(+)Sca-1(+)lin(-) starting cell population. However, compared with HSCs, the cytokine requirements for maximizing the generation of CFCs and total cells were at much lower cytokine doses. From the information provided by the factorial analysis, mathematical models based on Monod kinetics for inhibitory substrates were developed that allow total cell, CFC, and HSC expansion to be predicted as a function of the IL-11, SF, and FL concentrations in terms of more widely recognized parameters. Overall, these methods should also serve as a guide for the future design and testing of other ex vivo stem cell expansion systems.

Animals↗

PubMed2002

Controlled shear affinity filtration (CSAF): a new technology for integration of cell separation and protein isolation from mammalian cell cultures.

Controlled shear affinity filtration (CSAF) integrates animal cell separation and product isolation in a single unit operation through the use of a specifically designed rotating disk filter with incorporated membrane chromatography column. Because of the decoupling of shear force and pressure generation and the specific hydrodynamics of the system, shear rates can be easily optimized and precisely controlled to maximize filtration performance while viability of the shear sensitive animal cells is maintained. In this study, the general methodology is demonstrated using the integration of Chinese hamster ovary cell separation and isolation of recombinant tissue plasminogen activator (t-PA) as a model example. Direct capture of t-PA from cell culture broth was realized by using custom-made affinity membranes with lysine as a robust, small molecular weight affinity ligand. Small-scale t-PA adsorption experiments, as well as microfiltration experiments, were used to design the integrated CSAF process. A Chinese hamster ovary batch culture was processed with a lab-scale prototype, yielding 86% of the t-PA in the concentrated, particle-free eluate, whereas 95% of the bulk protein was removed. Because the viability of the cells is not significantly affected and high specific flux rates can be achieved, the CSAF technology should also be well suited for continuous perfusion with integrated product isolation. A truly continuous operation could be realized with two systems in tandem configuration.

Animals↗

PubMedDate not supplied

Optimization of an acoustic cell filter with a novel air-backflush system.

Increasing worldwide demand for mammalian cell production capacity will likely be partially satisfied by a greater use of higher volumetric productivity perfusion processes. An important additional component of any perfusion system is the cell retention device that can be based on filtration, sedimentation, and/or acoustic technologies. A common concern with these systems is that pumping and transient exposure to suboptimal medium conditions may damage the cells or influence the product quality. A novel air-backflush mode of operating an acoustic cell separator was developed in which an injection of bioreactor air downstream of the separator periodically returned the captured cells to the reactor, allowing separation to resume within 20 s. This mode of operation eliminated the need to pump the cells and allows the selection of a residence time in the separator depending on the sensitivity of the cell line. The air-backflush mode of operating a 10L acoustic separator was systematically tested at 10(7) cells/mL to define reliable ranges of operation. Consistent separation performance was obtained for wide ranges of cooling airflow rates from 0 to 15 L/min and for backflush frequencies between 10 and 40 h(-1). The separator performance was optimized at a perfusion rate of 10 L/day to obtain a maximum separation efficiency of 92 +/- 0.3%. This was achieved by increasing the power setting to 8 W and using duty cycle stop and run times of 4.5 and 45 s, respectively. Acoustic cell separation with air backflush was successfully applied over a 110 day CHO cell perfusion culture at 10(7) cells/mL and 95% viability.

Acoustics↗

PubMedDate not supplied

Logistic equations effectively model Mammalian cell batch and fed-batch kinetics by logically constraining the fit.

A four-parameter logistic equation was used to fit batch and fed-batch time profiles of viable cell density in order to estimate net growth rates from the inoculation through the cell death phase. Reduced three-parameter forms were used for nutrient uptake and metabolite/product formation rate calculations. These logistic equations constrained the fits to expected general concentration trends, either increasing followed by decreasing (four-parameter) or monotonic (three-parameter). The applicability of this approach was first verified for Chinese hamster ovary (CHO) cells cultivated in 15-L batch bioreactors. Cell density, metabolite, and nutrient concentrations were monitored over time and used to estimate the logistic parameters by nonlinear least squares. The logistic models fit the experimental data well, supporting the validity of this approach. Further evidence to this effect was obtained by applying the technique to three previously published batch studies for baby hamster kidney (BHK) and hybridoma cells in bioreactors ranging from 100 mL to 300 L. In 27 of the 30 batch data sets examined, the logistic models provided a statistically superior description of the experimental data than polynomial fitting. Two fed-batch experiments with hybridoma and CHO cells in benchtop bioreactors were also examined, and the logistic fits provided good representations of the experimental data in all 25 data sets. From a computational standpoint, this approach was simpler than classical approaches involving Monod-type kinetics. Since the logistic equations were analytically differentiable, specific rates could be readily estimated. Overall, the advantages of the logistic modeling approach should make it an attractive option for effectively estimating specific rates from batch and fed-batch cultures.

Animals↗