Comparison of a Molecular and an Immobilized Gadolinium(III)‐tris[(1R,4S)‐3‐heptafluorobutanoyl‐camphor] as Catalyst in the Asymmetric Danishefsky‐Hetero‐Diels‐Alder‐Reaction

On‐column reaction gas chromatography combines the power of separation and rapid analysis of reactants and reaction products with screening of reactions in a single step. Not only conversions but the reaction rates at various temperatures can be obtained from single measurements, making this approach superior to the time‐consuming measurements typically performed in reaction progress analysis. However, this approach has only been used in the investigation of interconversion processes, rearrangement reactions, and only a few examples of higher‐order reactions are known. Here we present the screening of immobilized gadolinium(III)‐tris[(1R,4S)‐3‐heptafluorobutanoyl‐camphor] in the Danishefsky‐hetero‐Diels‐Alder‐reaction by enantioselective on‐column reaction gas chromatography utilizing cryogenic focusing to achieve catalytic conversions in this higher‐order reaction and subsequent separation of the enantiomeric product mixture to determine the enantiomeric ratio. The results obtained by this approach could be transferred to the conventional batch reaction at a larger scale, demonstrating that on‐column reaction chromatography provides reliable results in the screening of enantioselective reactions. Chirality 26:243–248, 2014. © 2014 Wiley Periodicals, Inc.     

Enhancement of the antimicrobial properties of bacteriophage‐K via stabilization using oil‐in‐water nano‐emulsions

Bacteriophage therapy is a promising new treatment that may help overcome the threat posed by antibiotic‐resistant pathogenic bacteria, which are increasingly identified in hospitalized patients. The development of biocompatible and sustainable vehicles for incorporation of viable bacterial viruses into a wound dressing is a promising alternative. This article evaluates the antimicrobial efficacy of Bacteriophage K against Staphylococcus aureus over time, when stabilized and delivered via an oil‐in‐water nano‐emulsion. Nano‐emulsions were formulated via thermal phase inversion emulsification, and then bacterial growth was challenged with either native emulsion, or emulsion combined with Bacteriophage K. Bacteriophage infectivity, and the influence of storage time of the preparation, were assessed by turbidity measurements of bacterial samples. Newly prepared Bacteriophage K/nano‐emulsion formulations have greater antimicrobial activity than freely suspended bacteriophage. The phage‐loaded emulsions caused rapid and complete bacterial death of three different strains of S. aureus. The same effect was observed for preparations that were either stored at room temperature (18–20°C), or chilled at 4°C, for up to 10 days of storage. A response surface design of experiments was used to gain insight on the relative effects of the emulsion formulation on bacterial growth and phage lytic activity. More diluted emulsions had a less significant effect on bacterial growth, and diluted bacteriophage‐emulsion preparations yielded greater antibacterial activity. The enhancement of bacteriophage activity when delivered via nano‐emulsions is yet to be reported. This prompts further investigation into the use of these formulations for the development of novel anti‐microbial wound management strategies. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:932–944, 2014     

Advanced near‐infrared monitor for stable real‐time measurement and control of Pichia pastoris bioprocesses

Near‐infrared spectroscopy is considered to be one of the most promising spectroscopic techniques for upstream bioprocess monitoring and control. Traditionally the nature of near‐infrared spectroscopy has demanded multivariate calibration models to relate spectral variance to analyte concentrations. The resulting analytical measurements have proven unreliable for the measurement of metabolic substrates for bioprocess batches performed outside the calibration process. This paper presents results of an innovative near‐infrared spectroscopic monitor designed to follow the concentrations of glycerol and methanol, as well as biomass, in real time and continuously during the production of a monoclonal antibody by a Pichia pastoris high cell density process. A solid state instrumental design overcomes the ruggedness limitations of conventional interferometer‐based spectrometers. Accurate monitoring of glycerol, methanol, and biomass is demonstrated over 274 days postcalibration. In addition, the first example of feedback control to maintain constant methanol concentrations, as low as 1 g/L, is presented. Postcalibration measurements over a 9‐month period illustrate a level of reliability and robustness that promises its adoption for online bioprocess monitoring throughout product development, from early laboratory research and development to pilot and manufacturing scale operation. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:749–759, 2014     

SIP1 expression patterns in brain investigated by generating a SIP1‐EGFP reporter knock‐in mouse

A loss of function of SIP1 (Smad interacting protein 1) in the mouse as well as in human of Mowat–Wilson syndrome results in severe and multiple defects in neural tissue development, especially in the brain. However, no detailed expression analysis of SIP1 during brain development has been previously reported. In this study, we describe the generation of an EGFP knock‐in reporter mouse for the Sip1 locus and our subsequent analysis of SIP1‐EGFP fusion protein expression during brain development. SIP1‐EGFP expression was observed in the pyramidal neurons of the hippocampus, the dentate gyrus, and the postmitotic neurons in the cerebral cortex. In layer 5 of the cerebral cortex, SIP1‐EGFP expression was complementary to the Ctip2‐expressing neurons, most of which are thought to be the cortico‐spinal neurons. This suggested that SIP1‐EGFP expressing cells might have the specific trajectory targets other than the spinal region. We further observed SIP1‐EGFP expression in oligodendrocytes of the corpus callosum and fimbria, Bergmann glial cells of the cerebellum, the olfactory bulb, and in the serotonergic and dopaminergic neurons of the raphe nuclei in the brainstem. These findings may help to clarify the unknown roles of SIP1 in these cells and the pathoetiology of Mowat‐Wilson syndrome. genesis 52:56–67, 2014. © 2013 Wiley Periodicals, Inc.     

Mid‐infrared spectroscopy‐based analysis of mammalian cell culture Parameters

Within the framework of process analytical technology, infrared spectroscopy (IR) has been used for characterization of biopharmaceutical production processes. Although noninvasive attenuated total reflection (ATR) spectroscopy can be regarded as gold standard within IR‐based process analytics, simpler and more cost‐effective mid‐infrared (MIR) instruments might improve acceptability of this technique for high‐level monitoring of small scale experiments as well as for academia where financial restraints impede the use of costly equipment. A simple and straightforward at‐line mid‐IR instrument was used to monitor cell viability parameters, activity of lactate dehydrogenase (LDH), amount of secreted antibody, and concentration of glutamate and lactate in a Chinese hamster ovary cell culture process, applying multivariate prediction models, including only 25–28 calibration samples per model. Glutamate amount could be predicted with high accuracy (R² 0.91 for independent test‐set) while antibody concentration achieved good prediction for concentrations >0.4 mg L^?1. Prediction of LDH activity was accurate except for the low activity regime. The model for lactate monitoring was only moderately good and requires improvements. Relative cell viability between 20 and 95% could be predicted with low error (8.82%) in comparison to reference methods. An initial model for determining the number of nonviable cells displayed only acceptable accuracy and requires further improvement. In contrast, monitoring of viable cell number showed better accuracy than previously published ATR‐based results. These results prove the principal suitability of less sophisticated MIR instruments to monitor multiple parameters in biopharmaceutical production with relatively low investments and rather fast calibration procedures. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:578–584, 2015     

Rapid molecular methods for in‐field and laboratory identification of the yellow‐legged Asian hornet (Vespa velutina nigrithorax)

The yellow‐legged Asian hornet (Vespa velutina nigrithorax) is an invasive species that presents a threat to apiculture in Europe; first introduced into France in 2004, it has subsequently spread into neighbouring European countries. There is a risk of invasion and establishment in the UK, and in 2016, nests were found and destroyed in Alderney in the Channel Islands, and in Tetbury, Gloucestershire, illustrating a need for screening of suspect specimens so that invading hornets can be rapidly identified, and their nests destroyed. In this study, loop‐mediated isothermal amplification (LAMP) and real‐time PCR assays were developed to enable both in‐field and laboratory testing. Species‐specific identification assays and generic invertebrate control assays were developed. All the assays were validated according to the European Plant Protection Organisation standard PM 7/98. The assays were tested successfully against V. velutina nigrithorax obtained from France, Asia and the UK. Eight non‐target species, that were closely related or morphologically similar to the Asian hornet, gave negative results with the species‐specific assays, and positive results with the control assays. The assays could be used to detect target DNA at concentrations as low as 5 pg per reaction. LAMP was rapid, and cable of generating positive results within 10 min. Using simplified sample homogenization protocols that could be performed in the field, the LAMP assay was successful when tested against all developmental stages and nest samples, assisting with identification of samples that cannot be determined morphologically and allowing detection away from the laboratory. These assays provide a valuable tool for fast and reliable detection of this invasive species, offering the ability to identify damaged/incomplete specimens and immature life‐stages.     

Short‐ and long‐term effects on mAb‐producing CHO cell lines after cryopreservation

Cryopreservation provides the foundation for research, development, and manufacturing operations in the CHO‐based biopharmaceutical industry. Despite its criticality, studies are lacking that explicitly demonstrate that the routine cell banking process and the potential stress and damage during cryopreservation and recovery from thaw have no lasting detrimental effects on CHO cells. Statistics are also scarce on the decline of cell‐specific productivity (Q_p) over time for recombinant CHO cells developed using the glutamine synthetase (GS)‐based methionine sulfoximine (MSX) selection system. To address these gaps, we evaluated the impact of freeze‐thaw on 24 recombinant CHO cell lines (generated by the GS/MSX selection system) using a series of production culture assays. Across the panel of cell lines expressing one of three monoclonal antibodies (mAbs), freeze‐thaw did not result in any significant impact beyond the initial post‐thaw passages. Production cultures sourced from cryopreserved cells and their non‐cryopreserved counterparts yielded similar performance (growth, viability, and productivity), product quality (size, charge, and glycosylation distributions), and flow cytometric profiles (intracellular mAb expression). However, many production cultures yielded lower Q_p at increased cell age: 17 of the 24 cell lines displayed ≥20% Q_p decline after ～2–3 months of passaging, irrespective of whether the cells were previously cryopreserved. The frequency of Q_p decline underscores the continued need for understanding the underlying mechanisms and for careful clone selection. Because our experiments were designed to decouple the effects of cryopreservation from those of cell age, we could conclusively rule out freeze‐thaw as a cause for Q_p decline. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:463–477, 2018     

Case study and application of process analytical technology (PAT) towards bioprocessing: Use of tryptophan fluorescence as at‐line tool for making pooling decisions for process chromatography

Process analytical technology (PAT) has been gaining momentum in the biopharmaceutical community due to the potential for continuous real time quality assurance resulting in improved operational control and compliance. Two imperatives for implementing any PAT tool are that “variability is managed by the process” and “product quality attributes can be accurately and reliably predicted over the design space established for materials used, process parameters, manufacturing, environmental, and other conditions.” Recently, we have been examining the feasibility of applying different analytical tools to bioprocessing unit operations. We have previously demonstarted that commercially available online‐high performance liquid chromatography and ultra performance liquid chromatography systems can be used for analysis that can facilitate real‐time decisions for column pooling based on product quality attributes (Rathore et al., 2008 a,b). In this article, we review an at‐line tool that can be used for pooling of process chromatography columns. We have demonstrated that our tryptophan fluorescence method offers a feasible approach and meets the requirements of a PAT application. It is significantly faster than the alternative of fractionation, offline analysis followed by pooling. Although the method as presented here is not an online method, this technique may offer better resolution for certain applications and may be a more optimal approach as it is very conducive to implementation in a manufacturing environment. This technique is also amenable to be used as an online tool via front face fluorescence measurements done concurrently with product concentration determination by UV. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Rational Design of Hierarchical “Ceramic‐in‐Polymer” and “Polymer‐in‐Ceramic” Electrolytes for Dendrite‐Free Solid‐State Batteries

Solid polymer electrolytes as one of the promising solid‐state electrolytes have received extensive attention due to their excellent flexibility. However, the issues of lithium (Li) dendrite growth still hinder their practical applications in solid‐state batteries (SSBs). Herein, composite electrolytes from “ceramic‐in‐polymer” (CIP) to “polymer‐in‐ceramic” (PIC) with different sizes of garnet particles are investigated for their effectiveness in dendrite suppression. While the CIP electrolyte with 20 vol% 200 nm Li_6.4La₃Zr_1.4Ta_0.6O₁₂ (LLZTO) particles (CIP‐200 nm) exhibits the highest ionic conductivity of 1.6 × 10^?4 S cm^?1 at 30 °C and excellent flexibility, the PIC electrolyte with 80 vol% 5 µm LLZTO (PIC‐5 µm) shows the highest tensile strength of 12.7 MPa. A sandwich‐type composite electrolyte (SCE) with hierarchical garnet particles (a PIC‐5 µm interlayer sandwiched between two CIP‐200 nm thin layers) is constructed to simultaneously achieve dendrite suppression and excellent interfacial contact with Li metal. The SCE enables highly stable Li plating/stripping cycling for over 400 h at 0.2 mA cm^?2 at 30 °C. The LiFePO₄/SCE/Li cells also demonstrate excellent cycle performance at room temperature. Fabricating sandwich‐type composite electrolytes with hierarchical filler designs can be an effective strategy to achieve dendrite‐free SSBs with high performance and high safety at room temperature.     

A novel LED‐based 2D‐fluorescence spectroscopy system for in‐line bioprocess monitoring of Chinese hamster ovary cell cultivations—Part II

This study was performed in order to evaluate a new LED‐based 2D‐fluorescence spectrometer for in‐line bioprocess monitoring of Chinese hamster ovary (CHO) cell culture processes. The new spectrometer used selected excitation wavelengths of 280, 365, and 455 nm to collect spectral data from six 10‐L fed‐batch processes. The technique provides data on various fluorescent compounds from the cultivation medium as well as from cell metabolism. In addition, scattered light offers information about the cultivation status. Multivariate data analysis tools were applied to analyze the large data sets of the collected fluorescence spectra. First, principal component analysis was used to accomplish an overview of all spectral data from all six CHO cultivations. Partial least square regression models were developed to correlate 2D‐fluorescence spectral data with selected critical process variables as offline reference values. A separate independent fed‐batch process was used for model validation and prediction. An almost continuous in‐line bioprocess monitoring was realized because 2D‐fluorescence spectra were collected every 10 min during the whole cultivation. The new 2D‐fluorescence device demonstrates the significant potential for accurate prediction of the total cell count, viable cell count, and the cell viability. The results strongly indicated that the technique is particularly capable to distinguish between different cell statuses inside the bioreactor. In addition, spectral data provided information about the lactate metabolism shift and cellular respiration during the cultivation process. Overall, the 2D‐fluorescence device is a highly sensitive tool for process analytical technology applications in mammalian cell cultures.     

Members of the CIP4 family of proteins participate in the regulation of platelet‐derived growth factor receptor‐β‐dependent actin reorganization and migration

Background information. The F‐BAR {Fes/CIP4 [Cdc42 (cell division cycle 42)‐interacting protein 4] homology and BAR (Bin/amphiphysin/Rvs)} proteins have emerged as important co‐ordinators of signalling pathways that regulate actin assembly and membrane dynamics. The presence of the F‐BAR domain is the hallmark of this family of proteins and the CIP4 (Cdc42‐interacting protein 4) was one of the first identified vertebrate F‐BAR proteins. There are three human CIP4 paralogues, namely CIP4, FBP17 (formin‐binding protein 17) and Toca‐1 (transducer of Cdc42‐dependent actin assembly 1). The CIP4‐like proteins have been implicated in Cdc42‐dependent actin reorganization and in regulation of membrane deformation events visible as tubulation of lipid bilayers. Results. We performed side‐by‐side analyses of the three CIP4 paralogues. We found that the three CIP4‐like proteins vary in their effectiveness to catalyse membrane tubulation and actin reorganization. Moreover, we show that the CIP4‐dependent membrane tubulation is enhanced in the presence of activated Cdc42. Some F‐BAR members have been shown to have a role in the endocytosis of the EGF (epidermal growth factor) receptor and this prompted us to study the involvement of the CIP4‐like proteins in signalling of the PDGFRβ [PDGF (platelet‐derived growth factor) β‐receptor]. We found that knock‐down of CIP4‐like proteins resulted in a prolonged formation of PDGF‐induced dorsal ruffles, as well as an increased PDGF‐dependent cell migration. This was most likely a consequence of a sustained PDGFRβ activation caused by delayed internalization of the receptor in the cells treated with siRNA (small interfering RNA) specific for the CIP4‐like proteins. Conclusions. Our findings show that CIP4‐like proteins induced membrane tubulation downstream of Cdc42 and that they have important roles in PDGF‐dependent actin reorganization and cell migration by regulating internalization and activity of the PDGFRβ. Moreover, the results suggest an important role for the CIP4‐like proteins in the regulation of the activity of the PDGFRβ.     

Field evaluation on the synergistic attractiveness of 2‐(1‐undecyloxy)‐1‐ethanol and ipsenol to Monochamus saltuarius

To develop an optimal attractant for Monochamus saltuarius (Gebler) (Coleoptera: Cerambycidae), the synergistic effects of a few potential attractants (ethanol and α‐pinene as host‐plant volatiles, and ipsenol and ipsdienol as bark beetle pheromones) were tested in a pine forest combined with 2‐(1‐undecyloxy)‐1‐ethanol (monochamol), the aggregation pheromone of Monochamus species, for two consecutive years, 2014 and 2015. Total number of catches was 65 and 33 in 2014 and 2015, respectively. Ethanol or ethanol + monochamol (a base blend) were not attractive to M. saltuarius with no difference from the control. Addition of α‐pinene and ipsdienol to the base blend did not significantly increase catches. However, ipsenol was significantly synergistic to the base blend in attracting M. saltuarius in 2014, and the blend (ipsenol + base blend) attracted meaningfully higher numbers of M. saltuarius in 2015. Our study illustrates the potential for monochamol and ipsenol baits for monitoring and trapping of M. saltuarius in the field.     

Rapid optimization of protein freeze‐drying formulations using ultra scale‐down and factorial design of experiment in microplates

Retaining biopharmaceutical proteins in a stable form is critical to their safety and efficacy, and is a major factor for optimizing the final product. Freeze‐dried formulations offer one route for improved stability. Currently the optimization of formulations for freeze‐drying is an empirical process that requires many time‐consuming experiments and also uses large quantities of product material. Here we describe a generic framework for the rapid identification and optimization of formulation excipients to prevent loss of protein activity during a lyophilization process. Using factorial design of experiment (DOE) methods combined with lyophilization in microplates a range of optimum formulations were rapidly identified that stabilized lactose dehydrogenase (derived from Lactobacillus leichmanii) during freeze‐drying. The procedure outlined herein involves two rounds of factorially designed experiments—an initial screen to identify key excipients and potential interactions followed by a central composite face designed optimization experiment. Polyethylene glycol (PEG) and lactose were shown to have significant effects on maintaining protein stability at the screening stage and optimization resulted in an accurate model that was used to plot a window of operation. The variation of freezing temperatures and rates of sublimation that occur across a microplate during freeze‐drying have been characterized also. The optimum formulation was then freeze‐dried in stoppered vials to verify that the microscale data was relevant to the effects observed at larger pilot scales. This work provides a generic approach to biopharmaceutical formulation screening where possible excipients can be screened for single and interactive effects thereby increasing throughput while reducing costs in terms of time and materials. Biotechnol. Bioeng. 2009; 104: 957–964. © 2009 Wiley Periodicals, Inc.     

From water‐in‐oil to oil‐in‐water emulsions to optimize the production of fatty acids using ionic liquids in micellar systems

Biocatalysis is nowadays considered as one of the most important tools in green chemistry. The elimination of multiple steps involved in some of the most complex chemical synthesis, reducing the amounts of wastes and hazards, thus increasing the reaction yields and decreasing the intrinsic costs, are the major advantages of biocatalysis. This work aims at improving the enzymatic hydrolysis of olive oil to produce valuable fatty acids through emulsion systems formed by long alkyl chain ionic liquids (ILs). The optimization of the emulsion and the best conditions to maximize the production of fatty acids were investigated. The stability of the emulsion was characterized considering the effect of several parameters, namely, the IL and its concentration and different water/olive oil volumetric ratios. ILs from the imidazolium and phosphonium families were evaluated. The results suggest that the ILs effect on the hydrolysis performance varies with the water concentration and the emulsion system formed, that is, water‐in‐oil or oil‐in‐water emulsion. Although at low water concentrations, the presence of ILs does not present any advantages for the hydrolysis reaction, at high water contents (in oil‐in‐water emulsions), the imidazolium‐based IL acts as an enhancer of the lipase catalytic capacity, super‐activating 1.8 times the enzyme, and consequently promoting the complete hydrolysis of the olive oil for the highest water contents [85% (v/v)]. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1473–1480, 2015     

Implementation of proton transfer reaction‐mass spectrometry (PTR‐MS) for advanced bioprocess monitoring

We report on the implementation of proton transfer reaction‐mass spectrometry (PTR‐MS) technology for on‐line monitoring of volatile organic compounds (VOCs) in the off‐gas of bioreactors. The main part of the work was focused on the development of an interface between the bioreactor and an analyzer suitable for continuous sampling of VOCs emanating from the bioprocess. The permanently heated sampling line with an inert surface avoids condensation and interaction of volatiles during transfer to the PTR‐MS. The interface is equipped with a sterile sinter filter unit directly connected to the bioreactor headspace, a condensate trap, and a series of valves allowing for dilution of the headspace gas, in‐process calibration, and multiport operation. To assess the aptitude of the entire system, a case study was conducted comprising three identical cultivations with a recombinant E. coli strain, and the volatiles produced in the course of the experiments were monitored with the PTR‐MS. The high reproducibility of the measurements proved that the established sampling interface allows for reproducible transfer of volatiles from the headspace to the PTR‐MS analyzer. The set of volatile compounds monitored comprises metabolites of different pathways with diverse functions in cell physiology but also volatiles from the process matrix. The trends of individual compounds showed diverse patterns. The recorded signal levels covered a dynamic range of more than five orders of magnitude. It was possible to assign specific volatile compounds to distinctive events in the bioprocess. The presented results clearly show that PTR‐MS was successfully implemented as a powerful bioprocess‐monitoring tool and that access to volatiles emitted by the cells opens promising perspectives in terms of advanced process control. Biotechnol. Bioeng. 2012; 109: 3059–3069. © 2012 Wiley Periodicals, Inc.     

Green synthesis of enantiopure (S)‐1‐(benzofuran‐2‐yl)ethanol by whole‐cell biocatalyst

Optically active aromatic alcohols are valuable chiral building blocks of many natural products and chiral drugs. Lactobacillus paracasei BD87E6, which was isolated from a cereal‐based fermented beverage, was shown as a biocatalyst for the bioreduction of 1‐(benzofuran‐2‐yl) ethanone to (S)‐1‐(benzofuran‐2‐yl) ethanol with highly stereoselectivity. The bioreduction conditions were optimized using L. paracasei BD87E6 to obtain high enantiomeric excess (ee) and conversion. After optimization of the bioreduction conditions, it was shown that the bioreduction of 1‐(benzofuran‐2‐yl)ethanone was performed in mild reaction conditions. The asymmetric bioreduction of the 1‐(benzofuran‐2‐yl)ethanone had reached 92% yield with ee of higher than 99.9% at 6.73 g of substrate. Our study gave the first example for enantiopure production of (S)‐1‐(benzofuran‐2‐yl)ethanol by a biological green method. This process is also scalable and has potential in application. In this study, a basic and novel whole‐cell mediated biocatalytic method was performed for the enantiopure production of (S)‐1‐(benzofuran‐2‐yl)ethanol in the aqueous medium, which empowered the synthesis of a precious chiral intermediary process to be converted into a sophisticated molecule for drug production.     

Experimental conditions improving in‐solution target enrichment for ancient DNA

High‐throughput sequencing has dramatically fostered ancient DNA research in recent years. Shotgun sequencing, however, does not necessarily appear as the best‐suited approach due to the extensive contamination of samples with exogenous environmental microbial DNA. DNA capture‐enrichment methods represent cost‐effective alternatives that increase the sequencing focus on the endogenous fraction, whether it is from mitochondrial or nuclear genomes, or parts thereof. Here, we explored experimental parameters that could impact the efficacy of MYbaits in‐solution capture assays of ~5000 nuclear loci or the whole genome. We found that varying quantities of the starting probes had only moderate effect on capture outcomes. Starting DNA, probe tiling, the hybridization temperature and the proportion of endogenous DNA all affected the assay, however. Additionally, probe features such as their GC content, number of CpG dinucleotides, sequence complexity and entropy and self‐annealing properties need to be carefully addressed during the design stage of the capture assay. The experimental conditions and probe molecular features identified in this study will improve the recovery of genetic information extracted from degraded and ancient remains.     

Time‐lapsed imaging for in‐process evaluation of supercritical fluid processing of tissue engineering scaffolds

This article demonstrates the application of time‐lapsed imaging and image processing to inform the supercritical processing of tissue scaffolds that are integral to many regenerative therapies. The methodology presented provides online quantitative evaluation of the complex process of scaffold formation in supercritical environments. The capabilities of the developed system are demonstrated through comparison of scaffolds formed from polymers with different molecular weight and with different venting times. Visual monitoring of scaffold fabrication enabled key events in the supercritical processing of the scaffolds to be identified including the onset of polymer plasticization, supercritical points and foam formation. Image processing of images acquired during the foaming process enabled quantitative tracking of the growing scaffold boundary that provided new insight into the nature of scaffold foaming. Further, this quantitative approach assisted in the comparison of different scaffold fabrication protocols. Observed differences in scaffold formation were found to persist, post‐fabrication as evidenced by micro x‐ray computed tomography (μ x‐ray CT) images. It is concluded that time‐lapsed imaging in combination with image processing is a convenient and powerful tool to provide insight into the scaffold fabrication process. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009