In-situ microscopy: Online process monitoring of mammalian cell cultures

The in-situ microscope is a system developed to acquire images of mammalian cells directly inside a bioreactor (in-situ) duringa fermentation process. It requires only minimal operator intervention and it is well suited for either batch or long-termperfusion fermentation runs. The system fits into a 25 mm standard port and has a retractable housing, similar to the industry standard InTrac. Therefore, it can be cleaned and serviced without interruption of the process or risking contamination. A sampling zone inside the bioreactor encloses adefined volume of culture and an image sequence is taken. The height of the sampling zone is set by the control program and canbe adjusted during the cultivation to accommodate a wide range of change in cell density. The system has an infinity correctedoptical train and uses a progressive scan CCD camera to acquirehigh quality images. Process relevant information like cell density is extracted fromthe images by digital image processing software, currently in development for mammalian cells (CHO, BHK). The first version ofthe software will be able to estimate the cell density, cellsize distribution and to give information of the degree of aggregation (single and double cells, cell clusters).     

Automated flow cytometry for monitoring CHO cell cultures

Flow cytometry has been used to accurately monitor cell events that indicate the spatio-temporal state of a bioreactor culture. The introduction of process analytical technology (PAT) has led to process improvements using real-time or semi real-time monitoring systems. Integration of flow cytometry into an automated scheme for improved process monitoring can benefit PAT in bioreactor-based biopharmaceutical productions by establishing optimum process conditions and better quality protocols. Herein, we provide detailed protocols for establishing an automated flow cytometry system that can be used to investigate and monitor cell growth, viability, cell size, and cell cycle data. A method is described for the use of such a system primarily focused on CHO cell culture, although it is foreseen the information gathered from automated flow cytometry can be applied to a variety of cell lines to address both PAT requirements and gain further understanding of complex biological systems.     

Regulating apoptosis in mammalian cell cultures

Cell culture technology has become a widely accepted method used to derive therapeutic and diagnostic protein products. Mammalian cells adapted to grow in bioreactors now play an integral role in the development of these biologicals. A major limiting factor determining the output efficiency of mammalian cell cultures however, is apoptosis or programmed cell death. Methods to delay apoptosis and increase the longevity of cell cultures can lead to more economical processes. Researchers have shown that both genetic and chemical strategies to block apoptotic signals can increase cell culture productivity. Here, we discuss various strategies which have been implemented to improve cellular viabilities and productivities in batch cultures.     

Hybrid modeling framework for process analytical technology: application to Bordetella pertussis cultures

In the process analytical technology (PAT) initiative, the application of sensors technology and modeling methods is promoted. The emphasis is on Quality by Design, online monitoring, and closed-loop control with the general aim of building in product quality into manufacturing operations. As a result, online high-throughput process analyzers find increasing application and therewith high amounts of highly correlated data become available online. In this study, an hybrid chemometric/mathematical modeling method is adopted for data analysis, which is shown to be advantageous over the commonly used chemometric techniques in PAT applications. This methodology was applied to the analysis of process data of Bordetella pertussis cultivations, namely online data of near-infrared, (NIR), pH, temperature and dissolved oxygen, and off-line data of biomass, glutamate, and lactate concentrations. The hybrid model structure consisted of macroscopic material balance equations in which the specific reactions rates are modeled by nonlinear partial least square (PLS). This methodology revealed a significant higher statistical confidence in comparison to PLSs, translated in a reduction of mean squared prediction errors (e.g., individual root mean squared prediction errors calibration/validation obtained through the hybrid model for the concentrations of lactate: 0.8699/0.7190 mmol/L; glutamate: 0.6057/0.2917 mmol/L; and biomass: 0.0520/0.0283 OD; and obtained through the PLS model for the concentrations of lactate: 1.3549/1.0087 mmol/L; glutamate: 0.7628/0.3504 mmol/L; and biomass: 0.0949/0.0412 OD). Moreover, the analysis of loadings and scores in the hybrid approach revealed that process features can, as for PLS, be extracted by the hybrid method.     

Investigation of the potential of biocalorimetry as a process analytical technology (PAT) tool for monitoring and control of Crabtree-negative yeast cultures

Biological reaction calorimetry, also known as biocalorimetry, has led to extensive applications in monitoring and control of different bioprocesses. A simple real-time estimator for biomass and growth rate was formulated, based on in-line measured metabolic heat flow values. The performance of the estimator was tested in a unique bench-scale calorimeter (BioRC1), improved to a sensitivity range of 8 mW l^− 1 in order to facilitate the monitoring of even weakly exothermic biochemical reactions. A proportional–integral feedback control strategy based on these estimators was designed and implemented to control the growth rate of Candida utilis, Kluyveromyces marxianus and Pichia pastoris by regulating an exponential substrate feed. Maintaining a particular specific growth rate throughout a culture is essential for reproducible product quality in industrial bioprocesses and therefore a key sequence for the step from quality by analysis to quality by design. The potential of biocalorimetry as a reliable biomass monitoring tool and as a key part of a robust control strategy for aerobic fed-batch cultures of Crabtree-negative yeast cells in defined growth medium was investigated. Presenting controller errors of less than 4% in the best cases, the approach paves the way for the development of a generally applicable process analytical technology platform for monitoring and control of microbial fed-batch cultures.     

Apoptosis in CHO cell batch cultures: examination by flow cytometry

Chinese hamster ovary cells grown under conditions which are optimal for the production of a genetically engineered protein in batch culture, lose significant viability shortly after entering the stationary phase. This cell death was investigated morphologically and was found to be almost exclusively via apoptosi. Furthermore, cells were analyzed by flow cytometry using a fluorescent DNA end-labeling assay to label apoptotic cells, in conjunction with cell cycle analysis using propidium iodide. Apoptotic cells could be detected by this method, and by the radioactive end-labeling of extracted DNA, on all days of culture from day 1 to day 7; however, the degree of apoptotic cell death increased dramatically when the cells entered the stationary phase, rising to 50–60% of the total cell number at the termination of the culture. Flow cytometric analysis showed that the majority of cells underwent apoptosis whilst in G₁/G₀ and formed an apoptotic population with high DNA FITC end-labeling and hypodiploid propidium iodide binding. Additionally, the ability or inability to secrete specific protein products did not appear to interfere with the development of the apoptotic population with time.     

Application of thermal effusivity as a process analytical technology tool for monitoring and control of the roller compaction process

The aim of this study was to examine the relationship between physical characteristics of compacted ribbons and their thermal effusivity in an attempt to evaluate the feasibility of using effusivity for in-process monitoring of roller compaction. In this study, thermal effusivity, solid fraction, tensile strength, and Young's modulus of ribbons of microcrystal-line cellulose (MCC), anhydrous lactose, and placebo (PBO) formulations containing various ratios of MCC to anhydrous lactose (75∶20, 55∶40, 40∶55, and 20∶75) were determined at various compaction pressures (25–150 bars). The effusivity-square root of solid fraction relationship was linear for MCC and all the PBO formulations but was a second-order polynomial function for lactose. This could be due to the predominant deformation of lactose by brittle fracture, which might have significantly increased the number and size of contact points between particles, causing a change in thermal conductivity along with a density change. The effusivitytensile strength and effusivity-Young's modulus relationships were best described by logarithmic functions for MCC but were linear for lactose up to a compaction pressure of 65 bars. There were similar relationships for effusivity with tensile strength and Young's modulus for all PBO formulations except PBO IV, which might have been due to the deformation of lactose, the largest component in this formulation. Strong correlations between effusivity and physical properties of ribbons were established. Although these correlations were formulation-dependent, they demonstrate the possibility of using effusivity as a tool in monitoring roller compaction. Published: March 23, 2007     

Plug flow cytometry extends analytical capabilities in cell adhesion and receptor pharmacology

BACKGROUND: Plug flow cytometry is a recently developed system for the automated delivery of multiple small boluses or "plugs" of cells or particles to the flow cytometer for analysis. Important system features are that sample plugs are of precisely defined volume and that the sample vessel need not be pressurized. We describe how these features enable direct cell concentration determinations and novel ways to integrate flow cytometers with other analytical instruments. METHODS: Adhesion assays employed human polymorphonuclear neutrophils (PMNs) loaded with Fura Red and Chinese hamster ovary (CHO) cells cotransfected with genes for green fluorescent protein (GFP) and human P-selectin. U937 cells expressing the human 7-transmembrane formyl peptide receptor were loaded with the fluorescent probe indo-1 for intracellular ionized calcium determinations. A computer-controlled syringe or peristaltic pump loaded the sample into a sample loop of the plug flow coupler, a reciprocating eight-port valve. When the valve position was switched, the plug of sample in the sample loop was transported to the flow cytometer by a pressure-driven fluid line. RESULTS: In stirred mixtures of PMNs and CHO cells, we used plug flow cytometry to directly quantify changes in concentrations of nonadherent singlet PMNs. This approach enabled accurate quantification of adherent PMNs in multicell aggregates. We constructed a novel plug flow interface between the flow cytometer and a cone-plate viscometer to enable real-time flow cytometric analysis of cell-cell adhesion under conditions of uniform shear. The High Throughput Pharmacology System (HTPS) is an instrument used for automated programming of complex pharmacological cell treatment protocols. It was interfaced via the plug flow coupling device to enable rapid (< 5 min) flow cytometric characterization of the intracellular calcium dose-response profile of U937 cells to formyl peptide. CONCLUSIONS: By facilitating the coupling of flow cytometers to other fluidics-based analytical instruments, plug flow cytometry has extended analytical capabilities in cell adhesion and pharmacological characterization of receptor-ligand interactions.     

On-line monitoring of cell mass in mammalian cell cultures by acoustic densitometry

Acoustic resonance densitometry (ARD) provides a highly reproducible and stable method for on-line measurement of culture biomass density. The technique provides a direct determination of changes in relative density of culture medium and cell mass. At cell concentrations higher than 10(6) cells mL(-1)this method can replace cell counts and provide a continuous measure of total cell mass. In cultures of hybridomas or U937 human lymphoma cells, the ARD value correlates well with cell number except when the average cell size changes during culture. It is argued that cell mass determined by ARD rather than cell number should be used as the basis for measurements of specific biological activity.     

Real-time on-line flow cytometry for bioprocess monitoring

As the understanding of variation is the key to a good process and product quality one should pay attention to dynamics on the single-cell level. The basic idea of this approach was to qualify and quantify variations on the single-cell level during bioreactor cultivations by monitoring the expression of an eGFP tagged target protein (human membrane protein) using fully automated real-time, flow injection flow cytometry (FI-FCM). The FI-FCM system consists of a sampling- and defoaming- as well as of a dilution-section. It allows a very short monitoring interval (5 min) and is able to dilute the reactor sample by a factor ranging up to more than 10,000.In bioreactor cultivations of recombinant Pichia pastoris expressing the eGFP tagged target protein, high correlations (R² ≥ 0.97) between the FI-FCM fluorescent signal and other, however, population-averaged fluorescence signals (off-line fluorescence, in situ fluorescence probe) were obtained. FI-FCM is the only method able to distinguish between few cells with high fluorescence and many cells with low fluorescence intensity and proved that cells differ significantly from each other within the population during bioreactor cultivations. Single-cell fluorescence was distributed over a broad range within the cell population. These distributions strongly suggest that (a) the AOX-I promoter is leaky and (b) a fraction of the population is able to express more protein of interest within shorter time and (c) a fraction of the population does not express the fusion protein at all. These findings can help in the selection of high producing, stable strains. To show the platform-independency of the system, it has successfully been tested during bioreactor cultivations of three different strains (P. pastoris, Saccharomyces cerevisiae, Escherichia coli).Along with its applications in PAT, the FI-FCM could be used as a platform-independent (prokaryotes and eukaryotes) method in various other applications; for example in the closed-loop-control of bioprocesses using different kinds of fluorescent reporters, (waste- and drinking-) water analysis, clone selection in combination with FACS or even for surgery applications.     

Automated in-silico detection of cell populations in flow cytometry readouts and its application to leukemia disease monitoring

Background  

Identification of minor cell populations, e.g. leukemic blasts within blood samples, has become increasingly important in therapeutic disease monitoring. Modern flow cytometers enable researchers to reliably measure six and more variables, describing cellular size, granularity and expression of cell-surface and intracellular proteins, for thousands of cells per second. Currently, analysis of cytometry readouts relies on visual inspection and manual gating of one- or two-dimensional projections of the data. This procedure, however, is labor-intensive and misses potential characteristic patterns in higher dimensions.     

7-Aminoactinomycin D for apoptosis staining in flow cytometry

All species of the genus Rhodnius have a characteristic red coloration in their salivary glands due to the presence of heme proteins. Some of these secreted proteins, known as nitrophorins (NPs), are responsible for many of the antihemostatic activities of Rhodnius saliva such as anticoagulant and antihistamine. Several NPs have been described (NP1-4 and NP7), where NP7 is the only one with affinity to phospholipid membranes. Computational prediction suggested that NP7 also has an extended N-terminal tail on signal peptide cleavage; however, the complementary DNA does not allow the determination of the exact site of signal peptidase cleavage. On the other hand, according to previous studies, the exact length of the N-terminus has important consequences for the nitric oxide binding properties of NP7. Here, a method was developed to select phospholipid membrane-attaching proteins from homogenized tissue for analysis by mass spectrometry. The method was used to determine the exact N-terminus of the ferriheme protein NP7 from homogenates of the salivary glands of 5th instar nymphal stages of Rhodnius prolixus.     

Simplified off-gas analyses in animal cell cultures for process monitoring and control purposes

Batch-to-batch reproducibility of animal cell cultures can significantly be enhanced using process control procedures. Most informative signals for advanced process control can be derived from the volume fractions of oxygen and carbon dioxide in the vent line of the reactors. Here we employed simple low-cost sensors, previously not considered for off-gas analysis at a laboratory-scale cell cultures, and compared them with a simultaneously used quadrupole mass spectrometer, i.e., the standard equipment. A decisive advantage is that the sensors did not need any calibration and are easy to use. We show that monitoring and advanced control of cell cultures can significantly be simplified using the devices tested here and that the same batch-to-batch reproducibility can be obtained with much less effort than before.     

A flow injection flow cytometry system for on-line monitoring of bioreactors

For direct and on-line study of the physiological states of cell cultures, a robust flow injection system has been designed and interfaced with flow cytometry (FI-FCM). The core of the flow injection system includes a microchamber designed for sample processing. The design of this microchamber allows not only an accurate on-line dilution but also on-line cell fixation, staining, and washing. The flow injection part of the system was tested by monitoring the optical density of a growing E.coli culture on-line using a spectrophotometer. The entire growth curve, from lag phase to stationary phase, was obtained with frequent sampling. The performance of the entire FI-FCM system is demonstrated in three applications. The first is the monitoring of green fluorescent protein fluorophore formation kinetics in E.coli by visualizing the fluorescence evolution after protein synthesis is inhibited. The data revealed a subpopulation of cells that do not become fluorescent. In addition, the data show that single-cell fluorescence is distributed over a wide range and that the fluorescent population contains cells that are capable of reaching significantly higher expression levels than that indicated by the population average. The second application is the detailed flow cytometric evaluation of the batch growth dynamics of E.coli expressing Gfp. The collected single-cell data visualize the batch growth phases and it is shown that a state of balanced growth is never reached by the culture. The third application is the determination of distribution of DNA content of a S. cerevisiae population by automatically staining cells using a DNA-specific stain. Reproducibility of the on-line staining reaction shows that the system is not restricted to measuring the native properties of cells; rather, a wider range of cellular components could be monitored after appropriate sample processing. The system is thus particularly useful because it operates automatically without direct operator supervision for extended time periods.     

Modeling flow cytometry data for cancer vaccine immune monitoring

Flow cytometry (FCM) is widely used in cancer research for diagnosis, detection of minimal residual disease, as well as immune monitoring and profiling following immunotherapy. In all these applications, the challenge is to detect extremely rare cell subsets while avoiding spurious positive events. To achieve this objective, it helps to be able to analyze FCM data using multiple markers simultaneously, since the additional information provided often helps to minimize the number of false positive and false negative events, hence increasing both sensitivity and specificity. However, with manual gating, at most two markers can be examined in a single dot plot, and a sequential strategy is often used. As the sequential strategy discards events that fall outside preceding gates at each stage, the effectiveness of the strategy is difficult to evaluate without laborious and painstaking back-gating. Model-based analysis is a promising computational technique that works using information from all marker dimensions simultaneously, and offers an alternative approach to flow analysis that can usefully complement manual gating in the design of optimal gating strategies. Results from model-based analysis will be illustrated with examples from FCM assays commonly used in cancer immunotherapy laboratories.     

Multiparametric flow cytometric analysis of radiation-induced micronuclei in mammalian cell cultures.

A new flow cytometric method is presented that quantifies the frequency of radiation-induced micronuclei in mammalian cell cultures with high precision. After preparing a suspension of main nuclei and micronuclei stained with ethidium bromide and Hoechst 33258, both types of particles are measured simultaneously in a flow cytometer using forward light scatter and three fluorescence emission intensities excited by UV, 488 nm, and by energy transfer from Hoechst 33258 to ethidium bromide. Nonspecific debris overlapping the micronucleus distribution especially in the low fluorescence intensity region was discriminated from micronuclei by calculating ratios of the different fluorescences. The frequencies of radiation-induced micronuclei measured with this new technique agreed well with results obtained by conventional microscopy. The lower limit of the DNA content of micronuclei identified by this technique was found to be about 0.5%-0.75% of the DNA content of G1-phase nuclei. Dose effect curves and the time-dependent induction of micronuclei were measured for two different mouse cell lines.     

Defining viability in mammalian cell cultures

A large number of assays are available to monitor viability in mammalian cell cultures with most defining loss of viability as a loss of plasma membrane integrity, a characteristic of necrotic cell death. However, the majority of cultured cells die by apoptosis and early apoptotic cells, although non-viable, maintain an intact plasma membrane and are thus ignored. Here we measure the viability of cultures of a number of common mammalian cell lines by assays that measure membrane integrity (a measure of necrotic cell death) and assays that measure apoptotic cells, and show that discrepancies in the measurement of culture viability have a significant impact on the calculation of cell culture parameters and lead to skewed experimental data.