WO2002041021A1 - Dispositif pour l'analyse spectroscopique rmn in vivo des metabolites d'un milieu de culture - Google Patents
Dispositif pour l'analyse spectroscopique rmn in vivo des metabolites d'un milieu de culture Download PDFInfo
- Publication number
- WO2002041021A1 WO2002041021A1 PCT/FR2001/003567 FR0103567W WO0241021A1 WO 2002041021 A1 WO2002041021 A1 WO 2002041021A1 FR 0103567 W FR0103567 W FR 0103567W WO 0241021 A1 WO0241021 A1 WO 0241021A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- culture medium
- tubular
- conduits
- rectilinear
- arrangement
- Prior art date
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01R—MEASURING ELECTRIC VARIABLES; MEASURING MAGNETIC VARIABLES
- G01R33/00—Arrangements or instruments for measuring magnetic variables
- G01R33/20—Arrangements or instruments for measuring magnetic variables involving magnetic resonance
- G01R33/28—Details of apparatus provided for in groups G01R33/44 - G01R33/64
- G01R33/30—Sample handling arrangements, e.g. sample cells, spinning mechanisms
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01R—MEASURING ELECTRIC VARIABLES; MEASURING MAGNETIC VARIABLES
- G01R33/00—Arrangements or instruments for measuring magnetic variables
- G01R33/20—Arrangements or instruments for measuring magnetic variables involving magnetic resonance
- G01R33/44—Arrangements or instruments for measuring magnetic variables involving magnetic resonance using nuclear magnetic resonance [NMR]
- G01R33/46—NMR spectroscopy
- G01R33/465—NMR spectroscopy applied to biological material, e.g. in vitro testing
Definitions
- the present invention relates to a device for carrying out in vivo spectroscopic, qualitative and quantitative analyzes of metabolites of a cell culture medium, by nuclear magnetic resonance.
- It also relates to an assembly comprising a reactor containing the culture medium and such a device.
- Nuclear magnetic resonance spectroscopy a non-invasive and non-destructive technique, is the analytical tool best suited for in vivo analysis of intracellular metabolism.
- the two weak points of nuclear magnetic resonance technology in vivo reside on the one hand in the difficulty of reproducing in a measurement tube controlled conditions of the cellular medium, and on the other hand in its weak sensitivity (the signal / background noise is low).
- This signal / background noise ratio depends on the number of accumulations and therefore on the signal accumulation time and on the cell concentration in the measuring tube. The higher the signal accumulation time and / or the cell concentration, the greater this signal / background noise ratio.
- the rapidity with which the metabolism of a living system responds to a disturbance shows that it is not recommended to increase the accumulation time.
- Spectroscopic analysis of in vivo metabolism by nuclear magnetic resonance requires therefore a high cell density which accentuates the problems linked to the control of the physicochemical parameters of the cellular medium contained in the measurement tube and more particularly those linked to the oxygenation of the cells.
- the present invention provides a new device which makes it possible to carry out a continuous in vivo spectroscopic analysis of metabolites of a culture medium by nuclear magnetic resonance.
- the device comprises an arrangement of tubular conduits intended to be positioned in an apparatus for analysis by nuclear magnetic resonance, this arrangement of tubular conduits having between an inlet and an outlet suitable for being connected to a reactor containing the culture medium, a first part in which a helical path of the culture medium is defined during which the magnetization of the nuclei of the cells of the culture medium takes place, and a second part in which a rectilinear path is defined according to a direction and the opposite direction of the culture medium during which the analysis is carried out by nuclear magnetic resonance by excitation of the nuclei, a device in which means are provided for establishing, from the reactor, a continuous circulation of the culture medium between the inlet and outlet of said tubular duct arrangement.
- the means for circulating the culture medium in the arrangement of tubular conduits comprise a pump positioned between the reactor and the inlet of said device and having a flow rate of the order of 1 ml / s; the helical path defined in the first part of the arrangement of tubular conduits, extends over a determined length so that, at the flow rate of the selected pump, the travel time of said helical path through the culture medium is between approximately 3 and 5 times the longitudinal relaxation time T1 of the cell nuclei analyzed from the culture medium;
- the second part of the arrangement of tubular conduits has a measurement taking area by the nuclear magnetic resonance analysis apparatus, covering a part of the rectilinear outward and return path traveled by the culture medium; flexible plastic pipes are provided for the connection of the inlet and outlet of the device with the interior of the reactor containing the culture medium;
- the protective and holding part (130) is made of polyamide, and preferably of Plexiglas.
- the device comprises a first tubular conduit which has a substantially rectilinear primer open at one end to form the inlet of the device, then a spiral portion forming the helical path, followed by '' a rectilinear portion forming the straight rectilinear path which communicates with a second substantially rectilinear tubular conduit forming the rectilinear path of back and part of which extends through said spiral portion of the first tubular conduit to open through an opening forming the outlet of said device.
- the inlet and outlet sections of the first and second tubular conduits are identical.
- the second duct and the rectilinear portion of the first duct can be positioned concentrically or the rectilinear portion of the first tubular duct can be juxtaposed and parallel to a part of the second tubular duct, said conduits being connected by a bent bottom.
- the straight portion of the first tubular conduit and said portion of the second tubular conduit are formed by a single tube cut longitudinally by a central wall into two equal parts.
- the spiral part of the device according to the invention comprises ten turns.
- tubular conduits are made of glass, and the first and second conduits form a single piece.
- the invention also provides an assembly for the in vivo spectroscopic analysis of metabolites of a culture medium, by nuclear magnetic resonance, which comprises a device as mentioned above and a reactor containing the culture medium to be analyzed.
- This set includes a spectroscopic analysis apparatus by nuclear magnetic resonance specially adapted to receive said analysis device.
- FIG. 1 is a schematic side perspective view of a first embodiment of the device according to the invention.
- Figure 2 is a schematic view in longitudinal section of Figure 1;
- FIG. 3 is a schematic side view of a second embodiment of the device according to the invention.
- FIG. 5 shows the kinetics of the intensity of the H signal of methyl ethanol when increasing its concentration from 1 to 4 mM then from 4 to 20 mM during protocol test 1;
- FIG. 7 shows the kinetics of the intensity of the signal C of the methyl of ethanol during the increase of its concentration from 0.24 to 1M during the protocol test 2.
- FIGS. 1 to 3 show a device 100 for the in vivo spectroscopic analysis of the metabolites of a culture medium by nuclear magnetic resonance. It comprises an arrangement of tubular conduits intended to be positioned along the Z axis in an analysis apparatus by nuclear magnetic resonance, here not shown. This arrangement of tubular conduits has an inlet 109 and an outlet 115 intended to be connected by flexible conduits (not shown) made of plastic material, inside a reactor (not shown) containing the culture medium.
- the arrangement of tubular conduits shown in Figures 1 to 3 comprises between the inlet 109 and the outlet 115 of said device, a first part 101 in which there is defined a helical path of the culture medium during which the nuclei of the cells of the medium of culture are magnetized, and a second part 102 in which there is defined a rectilinear round-trip path along the Z axis (that is to say in one direction and the opposite direction) of the culture medium, at during which the excitation of the analyzed nuclei takes place.
- Means are also provided in this device 100 for establishing a continuous circulation of the culture medium withdrawn from the reactor, between the inlet 109 and the outlet 115 of said arrangement of tubular conduits.
- the device 100 comprises a first tubular conduit 110 which has a substantially rectilinear primer 110a open at one end to form the inlet 109 of the device, then a spiral portion 111 forming the helical path, followed by a rectilinear portion 112 along the Z axis, forming the straight rectilinear path, which communicates with a second tubular conduit 120 substantially rectilinear along the Z axis, forming the rectilinear return path and whose a part extends through said spiral portion 111 of the first tubular conduit, to open out through an opening 115 forming the outlet of said device.
- the inlet and outlet sections of the first and second tubular conduits 110, 120 are advantageously identical, which makes it possible in particular to avoid the formation of air bubbles in the culture medium which circulates in said conduits.
- the tubular conduits are made of glass and form a single piece.
- a portion 113 of the second conduit 120 and the straight portion 112 of the first conduit 110 are positioned concentrically so that they communicate through the bottom 112a of said straight portion 112 , said part 113 comprising an opening 114 opening at this level.
- the rectilinear portion 112 of the first tubular conduit 110 is juxtaposed and parallel to a part 113 of the second tubular conduit 120, said conduits 112,113 being connected by a bent bottom 112a.
- the straight portion 112 of the first tubular conduit 110 and said portion 113 of the second tubular conduit 120 are formed by a single tube cut longitudinally by a central wall into two equal parts communicating through the bottom 112a .
- the second part 102 of the arrangement of tubular conduits 110, 120 has an area for taking the measurement 103 by the nuclear magnetic resonance analysis apparatus, covering part of the rectilinear round trip traveled by the culture medium.
- the glass tubular conduits have an external diameter of approximately 4 mm and an internal diameter of approximately 3 mm, the external dimensions of the tubular conduits forming the second part 102 of the device are here of the order of 5 mm. so as to be specially adapted for a nuclear magnetic resonance spectrometer comprising a 5 mm probe and operating at a frequency of 500 MHz.
- the spiral portion of the first tubular conduit 111 has 10 turns and according to the examples of Figures 1 and 2, it has 8 turns.
- the means for circulating the culture medium in the arrangement of tubular conduits 110, 120 of the device 100 comprise a pump, not shown, positioned between the reactor and the inlet of said device and advantageously having a flow rate of the order of Iml / s.
- the number of turns is defined so that the helical path of the device extends over a determined length so that, at the flow rate of the pump chosen, the time of travel of said helical path through the culture medium is between approximately 3 and 5 times the relaxation time of the cell nuclei of the culture medium.
- the length along the Z axis of the spiral portion is here fixed at 40 mm and the length of the straight back and forth portion located in the extension along the Z axis of the spiral portion, is fixed at approximately 85 mm.
- the method of continuously and in vivo detection of metabolites of cells cultured in situ in a reactor using such a device 100 consists in: establish a circulation of said cells from the reactor to the device 100 positioned in the spectroscopic analysis apparatus by nuclear magnetic resonance, so that the nuclei of the circulating cellular medium are magnetized during the spiral path, and that excites certain nuclei and the nuclear magnetic resonance spectrum of these nuclei is recorded, then the spectrum recorded is compared to spectra obtained for conditions different from the culture medium in the reactor.
- the protocol tests are carried out according to the aforementioned device 100.
- the analyzes are carried out with a Bruker 500 spectrometer, with which the proton resonance frequency is 500 MHz.
- the assembly used is that presented in FIGS. 1 and 2.
- the total volume of the circuit is 79 ml, the flow rate of the pump of 0.5 ml / s.
- the total volume of liquid in the circuit is 100 ml.
- the average duration of the circuit is therefore 200 seconds.
- the inlet pipe into the reactor is placed above the liquid so as to obtain a drip, and the starting pipe is placed at the bottom of the reactor, so that the solution is homogenized in the reactor and that the ethanol concentration is uniform throughout the circuit after a few periods of 200 seconds.
- the homogenization of the magnetic field is optimized in the spectra recording area (area 103) using a 10% D 2 0 and 90% H 2 0 solution, without circulation of liquid. Then the circuit is emptied.
- the circuit contains 100 ml of a 1 mM aqueous ethanol solution.
- the test is repeated under the same conditions, except for the volume of the concentrated ethanol solution introduced into the reactor, such that the final concentration, after homogenization, throughout the circuit is 20 mM.
- the total kinetics of appearance of the CH 3 signal is represented in FIG. 5. It appears clearly in the figure the rapid initial increase in the concentration of ethanol at the level of the measurement probe, then after a few oscillations its stabilization.
- the experiment is repeated, initially with a 0.24 M ethanol solution in the circuit.
- a volume of concentrated ethanol solution is introduced, so that the final concentration, after homogenization throughout the circuit, is 1 M.
- 1D 13C spectra consisting of 64 acquisitions each composed of a preparation period of 10 ms duration followed by a high power pulse corresponding to an excitation of the C spins by 90 °, followed by an acquisition period of 590 ms duration, during which the scalar couplings
Landscapes
- Physics & Mathematics (AREA)
- Condensed Matter Physics & Semiconductors (AREA)
- General Physics & Mathematics (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
Claims
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU2002218370A AU2002218370A1 (en) | 2000-11-14 | 2001-11-14 | Device for spectroscopic nmr analysis of metabolites of a culture medium |
EP01996750A EP1342097A1 (fr) | 2000-11-14 | 2001-11-14 | Dispositif pour l'analyse spectroscopique rmn in vivo des metabolites d'un milieu de culture |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
FR0014609A FR2816713B1 (fr) | 2000-11-14 | 2000-11-14 | Dispositif pour l'analyse spectroscopique rmn in vivo des metabolites d'un milieu de culture |
FR00/14609 | 2000-11-14 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2002041021A1 true WO2002041021A1 (fr) | 2002-05-23 |
Family
ID=8856406
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/FR2001/003567 WO2002041021A1 (fr) | 2000-11-14 | 2001-11-14 | Dispositif pour l'analyse spectroscopique rmn in vivo des metabolites d'un milieu de culture |
Country Status (4)
Country | Link |
---|---|
EP (1) | EP1342097A1 (fr) |
AU (1) | AU2002218370A1 (fr) |
FR (1) | FR2816713B1 (fr) |
WO (1) | WO2002041021A1 (fr) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2011113980A1 (fr) * | 2010-03-16 | 2011-09-22 | Centro De Investigación Biomédica En Red En Bioingeniería, Biomateriales Y Nanomedicina (Ciber-Bbn) | Microchambre et dispositif de culture cellulaire pouvant être commandés par résonance magnétique nucléaire |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
ITPD20030142A1 (it) * | 2003-06-27 | 2004-12-28 | Livia Barenghi | Bioreattore, in particolare per spettroscopia nmr |
US7695957B2 (en) | 2005-07-22 | 2010-04-13 | Silvia Bradamante | Bioreactor, in particular for NMR spectroscopy |
DE102011082582B4 (de) * | 2011-09-13 | 2015-06-25 | Siemens Aktiengesellschaft | Verfahren zur Überwachung eines Fermentationsprozesses |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5313162A (en) * | 1991-04-27 | 1994-05-17 | Forschungszentrum Julich Gmbh | Apparatus for throughflow NMR spectroscopy, especially for cell suspensions |
-
2000
- 2000-11-14 FR FR0014609A patent/FR2816713B1/fr not_active Expired - Fee Related
-
2001
- 2001-11-14 EP EP01996750A patent/EP1342097A1/fr not_active Withdrawn
- 2001-11-14 WO PCT/FR2001/003567 patent/WO2002041021A1/fr not_active Application Discontinuation
- 2001-11-14 AU AU2002218370A patent/AU2002218370A1/en not_active Abandoned
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5313162A (en) * | 1991-04-27 | 1994-05-17 | Forschungszentrum Julich Gmbh | Apparatus for throughflow NMR spectroscopy, especially for cell suspensions |
Non-Patent Citations (2)
Title |
---|
J.F.HAW ET AL.: "Direct Coupling of a Liquid Chromatograph to a Continuous Flow Hydrogen Nuclear Magnetic Resonance Detector for Analysis of Petroleum and Synthetic Fuels", ANALYTICAL CHEMISTRY, vol. 52, no. 7, 1980, pages 1135 - 1140, XP001007509 * |
YUE ZHANG ET AL: "IMMOBILIZED FREE-RADICAL SUBSTRATES FOR MAGNETIZATION OF CARBON-13 NUCLEI IN FLOW NMR MEASUREMENTS", JOURNAL OF MAGNETIC RESONANCE,US,ACADEMIC PRESS, ORLANDO, FL, vol. 87, no. 1, 1 March 1990 (1990-03-01), pages 46 - 55, XP000132671, ISSN: 1090-7807 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2011113980A1 (fr) * | 2010-03-16 | 2011-09-22 | Centro De Investigación Biomédica En Red En Bioingeniería, Biomateriales Y Nanomedicina (Ciber-Bbn) | Microchambre et dispositif de culture cellulaire pouvant être commandés par résonance magnétique nucléaire |
ES2365282A1 (es) * | 2010-03-16 | 2011-09-28 | Centro De Investigacion Biomecanica En Red En Bioingenieria. Biomateriales Y Nanomedicina (Ciber-Bbn | Microcamara y dispositivo de cultivo celular monitorizables por resonancia magnetica nuclear. |
Also Published As
Publication number | Publication date |
---|---|
FR2816713B1 (fr) | 2003-04-11 |
AU2002218370A1 (en) | 2002-05-27 |
EP1342097A1 (fr) | 2003-09-10 |
FR2816713A1 (fr) | 2002-05-17 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CA1296692C (fr) | Rotor a pipetage dynamique pour dispositif d'analyse a centrifugation | |
EP2936145B1 (fr) | Dispositif et procede de discrimination d'un gaz dans un echantillon | |
Meinen et al. | Using a high finesse optical resonator to provide a long light path for differential optical absorption spectroscopy: CE-DOAS | |
EP1540363A1 (fr) | Methode de mesure de la mouillabilite de roches par resonance magnetique nucleaire | |
CN105259116A (zh) | 一种光声光谱痕量气体测量装置和方法 | |
EP0036801A1 (fr) | Appareil pour mesurer automatiquement la viscosité des liquides | |
WO2002041021A1 (fr) | Dispositif pour l'analyse spectroscopique rmn in vivo des metabolites d'un milieu de culture | |
EP3583403B1 (fr) | Procédé d'estimation de l'intensité d'une onde émise par une source émettrice | |
EP0035936A2 (fr) | Procédé et dispositif pour contrôler le fonctionnement d'une colonne d'extraction liquide-liquide | |
FR2491624A1 (fr) | Procede pour le fonctionnement d'un appareil d'analyse automatique | |
WO2012164229A1 (fr) | Porte-echantillon universel pour des mesures de caracteristiques electromagnetiques d'un materiau dielectrique et/ou magnetique | |
FR2726654A1 (fr) | Module fluidique pour dispositif d'analyse automatique | |
FR2649002A1 (fr) | Installation pour l'obtention par resonance magnetique nucleaire et echographie de donnees medicales, pharmacologiques ou autres | |
EP2294397A1 (fr) | Procédé et dispositif de détection de dépôts comportant au moins un matériau ferromagnétique sur ou à proximité de la paroi externe d'un tube | |
Smith et al. | Studies of vibrational relaxation in OCS and CF4 by pulsed photoacoustic techniques | |
EP3470821A1 (fr) | Dispositif photoacoustique multi-passage de detection de gaz | |
EP3593157A1 (fr) | Cellule de mesure par resonance magnetique nucleaire en milieu liquide avec une bobine à couplage inductif, systeme comprenant une telle cellule et son utilisation | |
EP3035070B1 (fr) | Cellule de mesure de rmn et ensemble de mesure de rmn | |
EP0589756B1 (fr) | Sonde pour la détection et le dosage d'oxygène moléculaire par spectrométrie de résonance paramagnétique électronique | |
EP0048531B1 (fr) | Dispositif de mesure de pressions en des endroits peu accessibles | |
FR3050529B1 (fr) | Dispositif et procede pour estimer ou mesurer la stabilite d'un melange par la variation dans la duree d'un signal electrique radiofrequence | |
FR2468120A1 (fr) | Cellule de mesure et dispositif automatique de microelectrophorese analytique | |
FR2952719A1 (fr) | Dispositif micro-ondes de mesure sans contact du taux d'humidite dans un materiau et procede associe | |
Lim et al. | Ultrafast Mid-IR Spectroscopy of Carbonmonoxymyoglobin: The Dynamics of Ligand Motion | |
Alrasheedi | Vibrational spectroscopic applications of Fourier transform infrared and Raman spectroscopy in biochemistry and microbiology |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
WWE | Wipo information: entry into national phase |
Ref document number: 2001996750 Country of ref document: EP |
|
WWP | Wipo information: published in national office |
Ref document number: 2001996750 Country of ref document: EP |
|
REG | Reference to national code |
Ref country code: DE Ref legal event code: 8642 |
|
WWW | Wipo information: withdrawn in national office |
Ref document number: 2001996750 Country of ref document: EP |
|
NENP | Non-entry into the national phase |
Ref country code: JP |
|
WWW | Wipo information: withdrawn in national office |
Country of ref document: JP |