Real time monitoring of multiple parameters in mammalian cell culture bioreactors using an in-line Raman spectroscopy probe

The FDA's process analytical technology initiative encourages drug manufacturers to apply innovative ideas to better understand their processes. There are many challenges to applying these techniques to monitor mammalian cell culture bioreactors for biologics manufacturing. These include the ability to monitor multiple components in complex medium formulations non-invasively and in-line. We report results that demonstrate, for the first time, the technical feasibility of the in-line application of Raman spectroscopy for monitoring a mammalian cell culture bioreactor. A Raman probe was used for the simultaneous prediction of culture parameters including glutamine, glutamate, glucose, lactate, ammonium, viable cell density, and total cell density.     

利用调制荧光仪在线监测叶绿素荧光

介绍了利用便携式调制荧光仪PAM-2100在线监测叶绿素荧光的技术。该技术不影响植物的自然光合状态,司以在线监测Ft、F′m、Y、rETR、qP、qN或NPQ、PAR和叶温等指标。以凤眼莲为例进行了在线监测,每隔5min监测一次,共进行了225min的监测。结果表明叶绿索荧光参数的变化依赖于PAR的变化。Ft、rETR、qN和NPQ的变化与PAR的变化趋势一致,F′m、Y和qP的变化与PAR的变化相反。通过对风眼莲的在线监测,说明该技术是可靠的,具有简单、快速、灵敏等特点。随着新型调制荧光仪的出现,该技术可能在植物生态学领域得到广泛应用。     

Single cell fluorescence imaging using metal plasmon-coupled probe

This work constitutes the first fluorescent imaging of cells using metal plasmon-coupled probes (PCPs) at single cell resolution. N-(2-Mercapto-propionyl)glycine-coated silver nanoparticles were synthesized by reduction of silver nitrate using sodium borohyride and then succinimidylated via ligand exchange. Alexa Fluor 647-labeled concanavalin A (con A) was chemically bound to the silver particles to make the fluorescent metal plasmon-coupled probes. The fluorescence images were collected using a scanning confocal microscopy. The fluorescence intensity was observed to enhance 7-fold when binding the labeled con A on a single silver particle. PCPs were conjugated on HEK 293 A cells. Imaging results demonstrate that cells labeled by PCPs were 20-fold brighter than those by free labeled con A.     

An online, non-invasive fluorescence probe for immobilized cell culture process development

An online, analytical technology was developed that utilized fluorescence to detect cells during an immobilized cell culture process. Chinese hamster ovary (CHO) cells that produced monoclonal antibodies (mAb) were transfected to express green fluorescent protein (GFP), and stable, fluorescence-positive cells were obtained by fluorescence-activated cell sorting (FACS). The immobilized cell culture process was then used to test the effects of sodium butyrate on cells. In this study, cells were cultured in porous, fibrous matrices that were placed in spinner flasks. A lab-scale, perfusion bioreactor with computer-controlled, online fluorescence sensors that continuously detected GFP fluorescence and quantified cell growth was utilized. In addition, the level of GFP fluorescence was used to predict mAb production in the culture without sampling for cell counting and protein analysis. Thus, non-invasive, fluorescence detection of cells provided a rapid, reliable and robust approach for developing an immobilized cell culture process.     

Plant cell wall characterization using scanning probe microscopy techniques

Lignocellulosic biomass is today considered a promising renewable resource for bioenergy production. A combined chemical and biological process is currently under consideration for the conversion of polysaccharides from plant cell wall materials, mainly cellulose and hemicelluloses, to simple sugars that can be fermented to biofuels. Native plant cellulose forms nanometer-scale microfibrils that are embedded in a polymeric network of hemicelluloses, pectins, and lignins; this explains, in part, the recalcitrance of biomass to deconstruction. The chemical and structural characteristics of these plant cell wall constituents remain largely unknown today. Scanning probe microscopy techniques, particularly atomic force microscopy and its application in characterizing plant cell wall structure, are reviewed here. We also further discuss future developments based on scanning probe microscopy techniques that combine linear and nonlinear optical techniques to characterize plant cell wall nanometer-scale structures, specifically apertureless near-field scanning optical microscopy and coherent anti-Stokes Raman scattering microscopy.     

Real-time fluorescence detection of multiple microscale cell culture analog devices in situ.

We investigated multiple microscale cell culture analog (microCCA) assays in situ with a high-throughput imaging system that provides quantitative, nondestructive, and real-time data on cell viability. Since samples do not move between measurements, captured images allow accurate time-course measurements of cell population response and tracking the fate of each cell type on a quantitative basis. The optical system was evaluated by measuring the short-term response to ethanol exposure and long-term growth of drug-resistant tumor cell lines with simultaneous samples. The optical system based on epi-fluorescent excitation consists of an LED and a CCD as well as discrete optical components for imaging a large number of cells simultaneously. HepG2/C3A and MESSA cell lines were cultured in two microCCA systems for continuous cell status monitoring in cell death experiments with ethanol and long-term cell growth. The experiment that tested ethanol uptake showed that ethanol immediately caused cell death. The system was applied to extracting dynamic constants in the uptake process. In the long-term cell growth experiment, growth of MESSA cells was followed by a stationary phase and eventual cell death attributed to nutrient and oxygen depletion and a change in the pH because of the accumulation of wastes by cell metabolism. HepG2/C3A cells were subject to contact inhibition and cell number did not change significantly over time. Issues related to long-term assays are also discussed. The quantitative results have been consistent with qualitative images and confirm the applicability of the portable optical system, and potential application to high-throughput analysis of cell-based assays to measure long-term dynamics.     

In situ monitoring of intracellular glucose and glutamine in CHO cell culture

The development of processes to produce biopharmaceuticals industrially is still largely empirical and relies on optimizing both medium formulation and cell line in a product-specific manner. Current small-scale (well plate-based) process development methods cannot provide sufficient sample volume for analysis, to obtain information on nutrient utilization which can be problematic when processes are scaled to industrial fermenters. We envision a platform where essential metabolites can be monitored non-invasively and in real time in an ultra-low volume assay in order to provide additional information on cellular metabolism in high throughput screens. Towards this end, we have developed a model system of Chinese Hamster Ovary cells stably expressing protein-based biosensors for glucose and glutamine. Herein, we demonstrate that these can accurately reflect changing intracellular metabolite concentrations in vivo during batch and fed-batch culture of CHO cells. The ability to monitor intracellular depletion of essential nutrients in high throughput will allow rapid development of improved bioprocesses.     

In situ monitoring of 3D in vitro cell aggregation using an optical imaging system

Bioreactor systems that maintain cells and tissues in suspension are increasingly popular for culturing 3D constructs to avoid the loss of in vivo cell function associated with traditional 2D culture methods. There is a need for the online monitoring of such systems to provide better understanding and control of the processes involved and to prevent the disruption of these processes caused by offline sampling and endpoint analysis. We describe a system for the imaging and analysis of cell aggregation, over long periods, within a high aspect rotating vessel (HARV). The system exploits side illumination, using an adjustable beam pattern, to restrict the detected light to that scattered by the cell aggregates, thus eliminating the need for the fluorescent labeling of the cells. The in situ aggregation of mammalian cells (MCF-7 breast carcinoma cells) was monitored over an 8 h period and image sequences showing the growth and motion of the aggregates within the bioreactor were obtained. Detailed size and population data have been derived characterizing the development of the aggregates during this time. We show how the number of resolvable aggregates increases to reach a peak and then declines as these aggregates merge. Once formed, remaining aggregates are found to consolidate to form more tightly packed bodies, typically reducing in cross-sectional area by one third. These results provide the basis for the development of an automated feedback system to control the growth of 3D cell cultures for repeatable, reliable, and quality controlled experimentation.     

In situ monitoring of cell death using Raman microspectroscopy

We investigated the use of Raman microspectroscopy to monitor the molecular changes in human lung carcinoma epithelial cells (A549) when cell death was induced by a toxic chemical. We treated A549 cells with 100 microM Triton X-100 and carried out Raman microspectroscopy measurements in parallel with cell viability and DNA integrity assays at time points of 0, 24, 48, and 72 hours. We found that the important biochemical changes taking place during cell death, such as the degradation of proteins, DNA breakdown, and the formation of lipid vesicles, can be detected with Raman microspectroscopy. A decrease in the intensity of the O-P-O stretching Raman peak corresponding to the DNA molecule phosphate-sugar backbone at 788 cm(-1) indicated DNA disintegration, an observation which was confirmed by DNA integrity analysis. We also found a decrease in the intensity of the Raman peaks corresponding to proteins (1005 cm(-1), 1342 cm(-1)) and an increase in the concentration of lipids (1660 cm(-1), 1303 cm(-1)). These changes are the effects of the complex molecular mechanisms during the induction of cell death, such as protein cleavage due to the activation of caspases, followed by DNA fragmentation.     

In situ fluorescence monitoring of immobilizedClostridium acetobutylicum

The NAD(P)H-dependent culture fluorescence of immobilizedClostridium acetobutylicum was followed during a three-part fermentation involving product formation on minimal and nitrogen-free media. The fluorescence signal, together with a knowledge of the metabolic pathways ofCl. acetobutylicum, provices information on the state and intracellular activities of the immobilized bacteria not readily found by other methods.     

Determination of Cryptosporidium parvum oocyst viability by fluorescence in situ hybridization using a ribosomal RNA-directed probe

AIMS: Fluorescence in situ hybridization (FISH) has been proposed for species-specific detection, and viability determination of Cryptosporidium parvum oocysts. FISH-based viability determination depends on rRNA decay after loss of viability. We examined the effects of RNase(s) and RNase inhibitors on FISH of C. parvum. METHODS AND RESULTS: FISH was performed using a 5'-Texas red-labelled DNA oligonucleotide probe at 1 pM microl(-1). Intact and heat-permeabilized oocysts were treated with 1-100 microg ml(-1) RNase. FISH of intact oocysts appeared unaffected by exogenous RNase if this was neutralized before permeabilization. FISH fluorescence of heat-killed oocysts stored in phosphate-buffered saline at room temperature decayed by 1/2 after 55 h, but remained detectable after 6 days. Addition of vanadyl ribonucleoside complex (VRC) extended rRNA half-life of heat-permeabilized oocysts to 155 h. CONCLUSIONS: Extended rRNA half-life may result in viability overestimation using FISH. RNase pretreatment before FISH is recommended to destroy residual rRNA in recently killed oocysts. Incorporation of 1-10 mM l(-1) VRC before FISH permeabilization steps should neutralize RNase activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Elimination of FISH fluorescence of nonviable C. parvum is desirable. Use of RNase and VRC is suggested to reduce numbers of false-positive 'viable' oocysts.     

Development of a fluorescence in situ hybridization method for cheese using a 16S rRNA probe

A 16S rRNA fluorescence in situ hybridization (FISH) method for cheese was developed to allow detection in situ of microorganisms within the dairy matrix. An embedding procedure using a plastic resin was applied to Stilton cheese, providing intact embedded cheese sections withstanding the hybridization reaction. The use of a fluorescein-labelled 16S rRNA Domain Bacteria probe allowed observation of large colonies of microbial cells homogeneously distributed in the cheese matrix. FISH experiments performed on cheese suspensions provided images of the different microbial morphotypes occurring. The technique has great potential to study the spatial distribution of microbial populations in situ in foods, especially where the matrix is too fragile to allow manipulation of cryosections.     

High-content proteomics: fluorescence multiplexing using an integrated,high-sensitivity,multiwavelength charge-coupled device imaging system

The detection of proteins in 2-D gels and their subsequent identification by MS is still the "gold standard" in proteomics. Fluorescent detection has increasingly replaced colorimetric and radiometric detection on gels and blots. The reasons for this are multiple and varied and include higher sensitivity, better quantitation, increased dynamic range, speed, safety and ease of use. Unlike other methods, fluorescent protein detection is also typically very consistent in response from protein to protein and in many cases is compatible with MS methods for protein identification. The superior sensitivity and benefits achieved by fluorescent techniques have spurred the development of instrumentation capable of delivering precise, sensitive, high-resolution image acquisition over a wide variety of excitation and emission wavelengths. This report focuses on applications using the highly sensitive, charge-coupled device based ProXPRESS multilabel imager, readily configurable for image acquisition over a wide variety of wavelengths (380-700 nm and ultraviolet (UV)) using xenon lamp or UV excitation. The ability to simultaneously detect enzyme activities or protein modifications with different color fluorescent probes in addition to total protein amounts (multiplexing) allows the further mining of proteomic data content from a single set of protein samples. To this end, the development of instrumentation that enables a multiplexing strategy will become central to in-depth proteomic studies. The ProXPRESS maximizes the efficiency of experimental strategies that require flexibility and multicolor fluorescence detection.     

In situ control of cell adhesion using photoresponsive culture surface

A photoresponsive culture surface (PRCS) allowing photocontrol of cell adhesion was prepared with a novel polymer material composed of poly(N-isopropylacrylamide) having spiropyran chromophores as side chains. Cell adhesion of the surface was drastically enhanced by the irradiation with ultraviolet (UV) light (wavelength: 365 nm); after subsequent cooling and washing on ice, many cells remained in the irradiated region, whereas most cells were removed from the nonirradiated region. The cell adhesion of the PRCS, which had been enhanced by previous UV irradiation, was reset by the visible light irradiation (wavelength 400-440 nm) and the annealing at 37 degrees C for 2 h. Also it was confirmed that the regional control of cell adhesion was induced several times by repeating the same series of operations. Further, living cell patterning with the 200 microm line width was produced readily by projecting UV light along a micropattern on the PRCS on which the living cells had been seeded uniformly in advance. By using a fluorescent probe that stains living cells only, it was confirmed that the cells maintained sufficient viability even after UV light irradiation followed by cooling and washing.     

Quantification of methanogens by fluorescence in situ hybridization with oligonucleotide probe

To monitor anaerobic environmental engineering system, new method of quantification for methanogens was tested. It is based on the measurement of specific binding (hybridization) of 16S rRNA-targeted oligonucleotide probe Arc915, performed by fluorescence in situ hybridization (FISH) and quantified by fluorescence spectrometry. Average specific binding of Arc915 probe was 13.4±0.5 amol/cell of autofluorescent methanogens. It was 14.3, 13.3, and 12.9 amol/cell at the log phase, at stationary phase and at the period of cell lysis of batch culture, respectively. Specific binding of Arc915 probe per 1 ml of microbial sludge suspension from anaerobic digester linearly correlated with concentration of autofluorescent cells of methanogens. Coefficient of correlation was 0.95. Specific binding of oligonucleotide probe Arc915 can be used for the comparative estimation of methanogens during anaerobic digestion of organic waste. Specific binding of Arc915 probe was linear function of anaerobic sludge concentration when it was between 1.4 and 14.0 mg/ml. Accuracy of the measurements in this region was from 5 to 12%.     

Using an online phycocyanin fluorescence probe for rapid monitoring of cyanobacteria in Macau freshwater reservoir

Monitoring of cyanobacteria and their toxins are traditionally conducted by cell counting, chlorophyll-a (chl-a) determination and cyanotoxin measurements, respectively. These methods are tedious, costly, time consuming, and insensitive to rapid changes in water quality and cyanobacterial abundance. We have applied and tested an online phycocyanin (PC) fluorescence probe for rapid monitoring of cyanobacteria in the Macau Storage Reservoir (MSR) that is experiencing cyanobacterial blooms. The relationships among cyanobacterial abundance, biovolume, cylindrospermopsin concentration, and PC fluorescence were analyzed using both laboratory and in-the-field studies. The performance of the probe was compared with traditional methods, and its advantages and limitations were assessed in pure and mixed cyanobacterial cultures in the laboratory. The proposed techniques successfully estimated the species including Microcystis and Cylindrospermopsis, two toxic species recently observed in the MSR. During February–November, 2010, the PC probe detected high correlations between PC and cell numbers (R ² = 0.71). Unlike the chl-a content, which indicates only the total algal biomass, the PC pigment specifically indicates cyanobacteria. These results support the PC parameter as a reliable estimate of cyanobacterial cell number, especially in freshwater bodies where the phytoplankton community and structure are stable. Thus, the PC probe is potentially applicable to online monitoring of cyanobacteria.     

A cell-based sensor system for toxicity testing using multiwavelength fluorescence spectroscopy

A novel cell-based fluorometric sensor system for toxicity monitoring is described, which uses functional spontaneously contracting cardiomyocytes (HL-1 cell line) as the biological recognition element. Based on these highly specialized cells, it has the potential of providing a sensitive and relevant analytical in vitro toxicity testing method. The system was configured by propagating the surface-attaching HL-1 cardiomyocytes in the wells of a 96-well microtiter plate and connecting the plate via an optical fiber to a fluorescence spectrometer capable of excitation-emission matrix scanning. The fluorescence data were analyzed using a conventional spectral analysis software program. The performance of the system for detection of general cytotoxicity to the cells was evaluated using three well-known drugs: verapamil, quinidine, and acetaminophen. The dose-response curves were assessed and the EC₅₀ values were determined (0.10 ± 0.007, 0.23 ± 0.025, and 12.32 ± 2.40 mM, respectively). Comparison with in vitro and in vivo reference data for the drugs showed good correlations, suggesting that this cell-based sensor system could be a useful tool in pharmacological in vitro drug testing.     

Plant cell culture initiation

The use of cultured plant cells in either organized or unorganized form has increased vey considerably in the last 10–15 yr. Many new technologies have been developed and applications in both fundamental and applied research have led to the development of some powerful tools for improving our knowledge of botanical systems and for gaining external influence over some of the key processes involved in inter-and intracellular organization. This is particularly the case when cell culture techniques are combined with those for the genetic modification of plant cells. Being able to regenerate whole plants that have gained or lost the expression of one or more specific genes has revolutionized the way in which we approach scientific questions and has opened up many additional possibilities for the molecular dissection of plants. The success or fall of all plant cell culture technologies lies with culture initiation. The choice of plant material, its physiologival state and cultivation history, the media used, and their means of preparation are just some of the factors that can greatly influence whether the desired end result will be achieved. In this article are described some of the practical aspects involved in successful plant cell culture initiation and the choices that have to be made. Attention is given to some of the pitfalls that can occur and how to avoid them. A good start is half the work     

In situ cell differentiation monitoring of Catharanthus roseus suspension culture processes by NIR spectroscopy

Bioprocess and Biosystems Engineering - Plant suspension culture is attracting interest as a promising platform to produce biological medicines due to the absence of virus, prions or DNA related to...