WO2011151148A2 - Procédé d'évaluation toxicologique, procédé de criblage toxicologique et système associé - Google Patents
Procédé d'évaluation toxicologique, procédé de criblage toxicologique et système associé Download PDFInfo
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- WO2011151148A2 WO2011151148A2 PCT/EP2011/057720 EP2011057720W WO2011151148A2 WO 2011151148 A2 WO2011151148 A2 WO 2011151148A2 EP 2011057720 W EP2011057720 W EP 2011057720W WO 2011151148 A2 WO2011151148 A2 WO 2011151148A2
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
- G01N33/5041—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects involving analysis of members of signalling pathways
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5082—Supracellular entities, e.g. tissue, organisms
- G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
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- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16C—COMPUTATIONAL CHEMISTRY; CHEMOINFORMATICS; COMPUTATIONAL MATERIALS SCIENCE
- G16C20/00—Chemoinformatics, i.e. ICT specially adapted for the handling of physicochemical or structural data of chemical particles, elements, compounds or mixtures
- G16C20/30—Prediction of properties of chemical compounds, compositions or mixtures
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- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16C—COMPUTATIONAL CHEMISTRY; CHEMOINFORMATICS; COMPUTATIONAL MATERIALS SCIENCE
- G16C20/00—Chemoinformatics, i.e. ICT specially adapted for the handling of physicochemical or structural data of chemical particles, elements, compounds or mixtures
- G16C20/70—Machine learning, data mining or chemometrics
Definitions
- the present invention relates to the field of bioreactors capable of simulating organs of living organisms such as mammalian organs, in particular human organs.
- the invention relates to the application of such bioreactors for the high-throughput screening of toxins on organs thus simulated.
- tissue or organ capable of mimicking the biological behavior of the organism.
- tissue or the corresponding organ called “natural” that is to say taken in its natural environment within the living organism
- These models are increasingly used in all phases of pharmaceutical research because they represent an advantageous alternative to in vivo models, ie to animal testing, against which both economic and ethical pressures appear. at an international level.
- the associated costs and logistics required for animal testing under conditions that comply with the French and European regulations on animal welfare and the recommendations of the competent health services, may make this technique prohibitive.
- the biological response (s) of the cell will therefore vary, sometimes considerably, depending on whether it is cultured in vitro or whether it is in the tissue or organ and, more generally, in the living organism.
- Petri dishes are cultured under "static" (non-fluid circulation) and non-"dynamic" conditions (with fluid circulation), which are the only ones likely to reflect the actual behavior of a tissue. or irrigated organ.
- NMR bioreactor development for live microbial functional analysis J Magn Reson 192 (1), 159-166 (2008), by Majors, PD, McLean, JS, & Scholten, JC, proposes a bioreactor located inside an NMR spectrometer (Nuclear Magnetic Resonance). NMR spectrometry makes it easy to analyze the culture medium of the bioreactor, but at the cost of very specific constraints. Indeed, the bioreactor is prohibited microstructures and materials other than glass, which limits its possibilities to those of petri dishes.
- the metabolomes present in the bioreactor undergo what is called a "derivatization", that is to say the targeted transformation of certain functional groups of the molecules to make them more volatile. And this greatly reduces the number of potentially detectable metabolites because it is restricted to the metabolites targeted by the derivatization.
- a third embodiment described in "High-throughput nuclear magnetic resonance metabolomic footprinting for tissue engineering," Tissue Eng Part C Methods 14 (2008), by Seagle, C, Christie, MA, Winnike, JH, McClelland, RE, Ludlow , JW, O'Connell, TM, Gamcsik, MP, & MacDonald, JM, consists of NMR measurement of metabolites derived from bioartificial organ culture media.
- the present invention aims to solve these difficulties by proposing a toxicological evaluation method on bioartificial tissues or organs perfectly simulating real organs, and proposing a new approach for analyzing the metabolic response.
- An object of the invention is to achieve this goal while providing a faster process and cheaper to implement, more reliable, providing results perfectly reproducible and much more easily exploitable.
- the present invention thus relates to a method of toxicological evaluation of a candidate substance on at least one tissue or organ, characterized in that it comprises the steps of:
- the invention proposes to study the indirect consequences of exposure of bioartificial organs to a toxic substance, by characterizing the metabolic signature of the response to this substance.
- the invention focuses on the use of "non-targeted” methods such as Nuclear Magnetic Resonance Spectroscopy (NMR) or mass spectrometry (MS), with or without chromatographic coupling, in order to detect as many metabolites as possible. endogenous.
- NMR Nuclear Magnetic Resonance Spectroscopy
- MS mass spectrometry
- chromatographic coupling In order to identify a global response at the metabolic scale, these different parallel measures are then analyzed by multidimensional statistics. These analyzes aim to "explain" the parameters of the experiment using the thousands of variables measured, and to deduce a prediction model. It is indeed possible to use supervised forms recognition techniques such as discriminant analysis, partial least squares regression (or PLS regression), artificial neural networks, and so on. According to other advantageous and nonlimiting features:
- step (d) is a partial least squares regression discriminant analysis (PLS-DA);
- step (d) is preceded by a step (d1) of locating and eliminating outliers in said multidimensional data set;
- step (d1) comprises a principal component unsupervised analysis (PCA);
- step (d) one of the statistical reliability estimation methods used in step (d) is cross-validation
- one of the statistical reliability estimation methods used in step (d) is a validation by the null hypothesis
- one of the statistical reliability estimation methods used in step (d) comprises calculating the area under a curve Receiver Operative Characteristic (ROC);
- the predetermined variables used in step (d) are chosen from the culture conditions, the cell type of the bioartificial fabric or organ, the type of said substance, and its dose;
- step (c) is carried out by at least one selected analytical chemistry technique, among the techniques of the group comprising proton NMR spectroscopy, carbon NMR spectroscopy, mass spectrometry, gas chromatography, liquid chromatography, and multiplexed detection methods;
- step (c) is carried out in the culture medium at the outlet of the bioreactor in order to observe the extracellular metabolic response
- step (c) is carried out on the cell pellets in order to observe the endogenous intracellular metabolic response
- the method further comprises a step (h) of obtaining a signature of specific toxicity of said substance from the list of biomarkers obtained in step (g);
- the method furthermore comprises a step (i) in which a bank of markers and / or of toxicity signatures is informed using the information obtained during the preceding steps.
- the present invention also aims to allow reliable toxicological screening at a very high rate, according to a second aspect.
- the present invention therefore also relates to a method for the toxicological screening of a candidate substance on at least one tissue or organ, characterized in that it comprises the steps of:
- said library of markers and / or signatures of toxicity has been established by the implementation of at least one method according to the first aspect of the invention.
- This high throughput screening of substances is made possible by the constitution of a database or database compiling the results of previous experiments.
- This application opens the door to the analytical screening of substances whose toxicity has never been demonstrated, by rapid, immediate and statistical comparison with reference substances, whose toxicity signature is known, that is to say a representation specific metabolic response.
- the invention finally relates to systems, one comprising at least one bioreactor, data processing means and a device for detecting biomarkers, characterized in that it is capable of implementing a toxicological evaluation method according to the first aspect of the invention, and the other further comprising data storage means, characterized in that it is capable of implementing a toxicological screening method according to the second aspect of the invention.
- FIG. 1 is an example of a 1 H NMR spectrum (proton NMR);
- FIG. 2 is a graph illustrating the result of a first experiment comparing a toxicological evaluation method according to the invention and a similar process that would use Petri dishes by modeling the 1 H NMR spectra by least squares regression. partial;
- FIG. 3 is a graph illustrating the result of a second experiment comparing different cell types by 1 H NMR and PLS regression;
- FIGS. 4a-c are three graphs illustrating the result of a third experiment comparing a metabolic response at different doses of the same xenobiotic by 1 H NMR analysis and PLS regression;
- FIGS. 5a-b are two examples of Receiver Operating Characteristic curves used in one embodiment of the toxicological evaluation method according to the invention.
- the toxicological evaluation method begins with a step of obtaining an artificial tissue or organ, on which the substance will be tested.
- bioreactors are used. These advantageously comprise a culture chamber comprising a microstructured upper wall and a lower wall promoting cell development, a fluid entry point and a fluid outlet point to allow the passage of a nutrient fluid necessary for the development and cell growth and ultimately exposure to the substance whose toxicological properties are to be studied.
- Such bioreactors are described in detail in the patent application FR0954288 filed June 23, 2009, which is referred to herein. The invention is however not limited to this type of bioreactor in particular.
- the microstructures of a bioreactor make possible the development of bioartificial tissues or organs presenting an elaborate cellular structure in conformity with reality. Growth is accelerated and autonomous. Bioreactors are easily mounted in series or in parallel. It is thus possible to cultivate following different families of cells, which together simulate an organ. Living organs are indeed very complex systems that generally house one main cell type, plus many others, whose presence is essential. For example, in the case of the liver 80% of the cells are what are called hepatocytes. But there are also endothelial cells, Kuppfer cells, Ito cells, hepatocyte lymphocytes ...
- the dynamic operation of the bioreactor makes it possible, in addition to a better simulation of the tissue or the organ, to expose to a substance and then to easily recover the metabolic response of the cells cultured at the level of the output connectors of the bioreactor. In this case, the so-called extracellular metabolic response is observed. Alternatively it may be useful to observe the endogenous metabolic response, i.e., within the cells themselves. In this case, the metabolic response is recovered directly on the cell pellets.
- bioartificial tissues or organs are thus cultivated until maturity, then exposed to a substance to be tested. After a predetermined time according to the test protocol, samples are recovered, whether it is output inside the cells.
- NMR nuclear magnetic resonance
- mass spectrometry a method of spectroscopy applied to a particle or a set of particles.
- NMRs based on atoms characteristics of organic molecules, such as hydrogen or carbon are particularly applicable to the invention.
- acquisition "without a priori" of a dataset associated with the metabolic response it will include acquisition of any data associated with any metabolic response, and not only data associated with "expected" elements of a metabolic response. No hypothesis concerning the metabolites involved in the biological or toxicological response of interest is to be made. In other words, an acquisition without a priori of a dataset associated with the metabolic response is an acquisition that incorporates including data corresponding to unknown metabolites. An unbiased approach differs fundamentally from the targeted metabolism approach in which anything that does not correspond to the targeted metabolites is ignored.
- NMR nuclear magnetic resonance
- spectroscopy allows an acquisition without a priori, unlike the use for example of a DNA chip, which limits the acquisition of data associated with the expression of the only genes in the chip, in other words, a partial metabolic response.
- Multidimensional statistics can then be used to identify significantly affected metabolic signals and in what proportions. Consequently, the a priori approach is of great interest for discovering new markers associated with this toxicological response. In the targeted approach, the potential for discovering new markers is non-existent.
- 1 H NMR proton NMR, which allows the detection of hydrogen atoms
- spectroscopy is used, with which a series of tests have been carried out.
- These tests involved two cell lines and three potentially toxic substances, both lines possibly being co-cultured.
- These cells are on the one hand HEPG2 / C3A, simply named C3A, human hepatocyte cells, and on the other hand MDCK (Madin-Darby Canine Kidney Cells), kidney cells of the dog.
- MDCK Medin-Darby Canine Kidney Cells
- the invention is in no way limited to liver or kidney-type bioartificial organs, and that one skilled in the art can transpose the invention to any type of bioartificial organ that can be simulated or modeled in a bioreactor. In particular, mention may be made of the pancreas, the heart, the testicles, parts of the brain, etc.
- the xenobiotics to which the cells used in the tests were exposed are ammonia (NH3), dimethylsulfoxide (DMSO) and N-acetyl-para-aminophenol (APAP), better known as paracetamol. These three substances are known to have harmful effects on the liver at high doses.
- the medium samples are prepared, for example, using 350 ⁇ L of medium, mixed with 200 ⁇ l of 0.9 g / L saline solution composed of 90% water (H 2 O) and 10% heavy water (D 2 O). calibration purposes.
- the 550 L of solution are then transferred to an NMR analysis tube.
- NMR acquisition is done at a frequency of 700 MHz using a proton probe.
- 1 D a NMR spectrum is recorded using a presaturation of the water resonance in the following sequence: T rd -P 90 - ti-t-m P9o -P9o- aq.
- Trd represents a delay of 2s during which the resonance of the water is selectively irradiated
- P90 is a radio frequency pulse of 90 °
- t1 corresponds to a delay of 3 ⁇ .
- interferograms are multiplied by an exponential function corresponding to a line broadening of 0.3 Hz.
- the width of the interferogram is doubled by adding null points at the end of the interferogram, so as not to degrade the resolution of the spectrum.
- the scale in the horizontal dimension corresponds to chemical shifts 1 H. It extends over a window of 10 ppm around the resonance of water.
- the scale in the vertical dimension corresponds to the intensity of the resonances which is proportional to the number of protons present for a chemical group of each molecule of the sample.
- the spectra are then phased and a baseline linear correction is performed, before calibration on the signal of the beta anomer of glucose at 5.23ppm. The region between 4.67ppm and 5ppm around the residual water signal is suppressed for better visibility, and the spectra are exported as a multi-dimensional data set.
- FIG. 1 shows an example of a 1 H NMR spectrum of a sample of culture supernatant of C3A cells exposed to ammonia (25 ⁇ M / ⁇ M, 10 mM NH 3 ). The region comprising the aromatic resonances (between 6.5 and 10 ppm) has been enlarged. The peaks of the main metabolites are annotated by way of example.
- the statistical analysis consists first of all of an unsupervised principal component analysis to identify the outliers in the multidimensional dataset.
- the principal component analysis compresses the redundant 40,000 NMRs present in the dataset into a new orthonormal frame, each axis representing a major component of the data set variance, which is therefore multidimensional.
- a multivariate statistical analysis, called supervised, is then carried out in order to discriminate culture conditions or groups of doses, and identify specific metabolic signatures for toxicological effects.
- This supervised analysis is mainly performed using partial least squares (Partial Least Squares) regression, using Partial Least Squares (PLS-DA) discriminant analysis, but not limited to.
- a PLS regression just like the linear regression, makes it possible to identify the components of the dataset (explanatory variables, X) which are quantitatively correlated with the variable to be explained (Y), whether they are culture conditions, the cell type of the bioartificial organ, the type of treatment administered or the dose of the treatment.
- Artificial neural network structures can also be programmed to perform this multivariate analysis.
- the coordinates of the samples of the test set are then calculated, which makes it possible to assign a prediction (dose or group) to these test samples, and to calculate the prediction error.
- the prediction error may be in the form of a confusion matrix for calculating an error rate (or good prediction), or in the form of calculating an error coefficient, typically, Q2 .
- a second step it is also possible to test the robustness of the model by randomly generating a series of data sets for which the variable to explain Y has been permuted. There is thus no more relation between the variable to be explained Y and the explanatory variables X, which corresponds in statistics to validation by the null hypothesis.
- a series of several hundred models following the null hypothesis is generated and their robustness is evaluated by cross validation. It is then possible to show that the more random the models, the more difficult it is to build a predictive model. We can then show that the initial model is significantly different from the population of random models following the null hypothesis, which validates all the more strongly the initial model.
- ROC Operating Characteristic of a receiver
- a ROC curve gives the rate of correct classifications in a group (called true positive rate) according to the number of incorrect classifications (false positive rate) for the same group.
- the curve is therefore included in a square of side 1, and necessarily passes through (0,0) and (1, 1).
- a biomarker identification step follows. It makes it possible to better reflect the reality of the metabolic response, since it makes it possible to find the metabolites associated with the components of the dataset selected for the model, that is to say the most characteristic of the exposure to the toxic substance. . This step is done for example by reading by a professional of the associated peaks on the NMR spectrum.
- signatures are stored in a database in order to constitute a signature bank.
- a marker library can be considered, each biomarker being referenced by the list of substances in which it has been involved.
- Such a bank may be very useful for another aspect of the invention which will be described below.
- the metabolic signature of a bioartificial organ culture in a bioreactor was compared to that of a conventional culture in Petri dishes.
- the metabolism of C3A bioartificial liver in a bioreactor was compared to that of C3A cells in conventional culture in Petri dishes.
- Two partial least squares discriminant analyzes (PLS-DA) were constructed to compare the culture of C3A cells in Petri dishes with that of C3A cells in a bioreactor with an optimal flux of 10 ⁇ l / min (Fig.2).
- PLS-DA models show clear discrimination between cells grown in Petri dishes and within bioreactors, demonstrating an endogenous metabolic signature of bioartificial organs, directly related to bioreactor culture.
- the coefficients of each PLS model were analyzed to identify the metabolites significantly affected by the bioreactor culture.
- Figure 2 also illustrates this result by showing that Petri dishes and bioreactors have different metabolic signatures.
- the cloud graph of the individuals shows a significant difference between the two families. Metabolite sign of correlation in
- C3A cells liver cells
- MDCK cells kidney cells
- C3A / MDCK liver-kidney co-culture
- PLS-DA three partial least squares discriminant analyzes were constructed to compare C3A bioartificial liver cultures with MDCK bioartificial protein kidney cultures and C3A / MDCK co-cultures with an optimal flux of 10 ⁇ L / min (Fig. 3).
- PLS-DA models show a clear segregation between the 3 types of organs. The coefficients of each PLS model were analyzed to identify significantly different metabolites between bioartificial organs.
- PLS regression models show a quantitative dose-response relationship, demonstrating an indirect endogenous metabolic response quantitatively related to the dose of NH3 administered.
- the coefficients of each PLS model were analyzed to identify the metabolites significantly affected by the NH3 dose. Comparing the lists of metabolites for each bioartificial organ shows that each one presents a unique and characteristic metabolic response, related to the physiology and cellular toxicology specific to each organ.
- PLS regression models show a quantitative dose-response relationship, demonstrating an indirect endogenous metabolic response of bioartificial livers quantitatively related to each treatment administered.
- the coefficients of each PLS model were analyzed to identify liver endogenous metabolites significantly affected by one treatment or another.
- the comparison of the metabolite lists for each treatment shows that the bioartificial liver has a unique metabolic response characteristic of each toxic molecule administered (Table 4). In this table, for each metabolite is indicated in parentheses the sign of the variation of its concentration when the dose of the xenobiotic of the experiment increases.
- ROC curves were calculated for several models to evaluate their performance.
- the toxicity predictions were for example made for a game exposed to APAP at 10 mM, and NH3 at 5 m.
- the ROC curves obtained are respectively represented by FIGS. 5a and 5b.
- the AUC is 0.943723 and 0.9335968 respectively, a confidence of nearly 95%.
- a 10mM NH3 toxicity assessment model even reached an AUC of 0.9994318 (99.94% confidence).
- the invention proposes a method for toxicological screening.
- the idea is to build a base of signatures by comparison with which we will be able very quickly to identify a xenobiotic.
- each time a new substance is evaluated with the method according to the first aspect its signature is added to a database. As a database becomes more and more complete appears.
- a tolerance threshold is set by the user.
- a very low tolerance level increases the probability of not detecting a known substance, but ensures a high degree of confidence if the substance is actually identified.
- a lower tolerance level makes it possible to propose several solutions, and to let the user conclude. Alternatively, it is not the signatures that are compared, but the lists of biomarkers detected, or directly the spectra.
- the invention proposes systems implementing the toxicological evaluation method according to the first aspect of the invention, or implementing the toxicological screening method according to the second aspect of the invention.
- These systems comprise at least one bioreactor, data processing means and a biomarker detection device.
- the system for the toxicological screening also has data storage means, on which will be stored the database containing for example the signatures of known xenobiotics.
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US13/696,861 US9448226B2 (en) | 2010-05-12 | 2011-05-12 | Method of toxicological evaluation, method of toxicological screening and associated system |
JP2013509573A JP5937576B2 (ja) | 2010-05-12 | 2011-05-12 | 毒性評価法、毒物スクリーニング法及びそのシステム |
EP11719026.4A EP2569628B8 (fr) | 2010-05-12 | 2011-05-12 | Procédé d'évaluation toxicologique, procédé de criblage toxicologique et système associé |
CA2799229A CA2799229C (fr) | 2010-05-12 | 2011-05-12 | Procede d'evaluation toxicologique, procede de criblage toxicologique et systeme associe |
IL222978A IL222978B (en) | 2010-05-12 | 2012-11-11 | Toxicological evaluation method, toxicological sorting method and system accordingly |
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FR954288A (fr) | 1947-10-15 | 1949-12-21 | Perfectionnements aux emballages étanches |
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CHRYSANTHOPOULOS, P.K., GOUDAR, C.T., KLAPA, M.1: "Metabolomics for high-resolution monitoring of the cellular physiological state in cell culture engineering", METAB ENG, 2009 |
MCLEAN, J.S., SCHOLTEN, J.C.: "NMR bioreactor development for live in-situ microbial functional analysis", J MAGN RESON, vol. 192, no. 1, 2008, pages 159 - 166, XP022635654, DOI: doi:10.1016/j.jmr.2008.02.014 |
SEAGLE, C., CHRISTIE, M.A., WINNIKE, J.H., MCCLELLAND, R.E., LUDLOW, J.W., O'CONNELL, T.M., GAMCSIK, M.P., MACDONALD, J.M.: "High- throughput nuclear magnetic resonance metabolomic footprinting for tissue engineering", TISSUE ENG PART C METHODS, vol. 14, 2008, XP002614422, DOI: doi:10.1089/ten.tec.2007.0401 |
Also Published As
Publication number | Publication date |
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FR2960062A1 (fr) | 2011-11-18 |
EP2569628A2 (fr) | 2013-03-20 |
US9448226B2 (en) | 2016-09-20 |
IL222978B (en) | 2018-11-29 |
EP2569628B8 (fr) | 2019-04-24 |
IL222978A0 (en) | 2013-02-03 |
FR2960062B1 (fr) | 2012-12-28 |
JP2013533734A (ja) | 2013-08-29 |
US20130060550A1 (en) | 2013-03-07 |
CA2799229C (fr) | 2020-02-11 |
EP2569628B1 (fr) | 2018-11-07 |
WO2011151148A3 (fr) | 2013-11-28 |
CA2799229A1 (fr) | 2011-12-08 |
JP5937576B2 (ja) | 2016-06-22 |
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