+

WO2001048144A2 - Chambre de culture de cellules mammaliennes - Google Patents

Chambre de culture de cellules mammaliennes Download PDF

Info

Publication number
WO2001048144A2
WO2001048144A2 PCT/US2000/041800 US0041800W WO0148144A2 WO 2001048144 A2 WO2001048144 A2 WO 2001048144A2 US 0041800 W US0041800 W US 0041800W WO 0148144 A2 WO0148144 A2 WO 0148144A2
Authority
WO
WIPO (PCT)
Prior art keywords
cell
membrane
cells
cell culture
impedance
Prior art date
Application number
PCT/US2000/041800
Other languages
English (en)
Other versions
WO2001048144A3 (fr
Inventor
Hal C. Cantor
Original Assignee
Advanced Sensor Technologies, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Advanced Sensor Technologies, Inc. filed Critical Advanced Sensor Technologies, Inc.
Priority to AU55155/01A priority Critical patent/AU5515501A/en
Publication of WO2001048144A2 publication Critical patent/WO2001048144A2/fr
Publication of WO2001048144A3 publication Critical patent/WO2001048144A3/fr
Priority to US10/129,248 priority patent/US6852525B1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M37/00—Means for sterilizing, maintaining sterile conditions or avoiding chemical or biological contamination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/10—Petri dish
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/24—Gas permeable parts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/38—Caps; Covers; Plugs; Pouring means
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/46—Means for regulation, monitoring, measurement or control, e.g. flow regulation of cellular or enzymatic activity or functionality, e.g. cell viability

Definitions

  • the present invention relates to a cell culture chamber 10. More
  • the present invention relates to a cell culture chamber 10 utilizing a
  • Biosensors sensors including biological materials employed to determine whether biological materials were used to determine whether biological materials were used to determine whether biological materials were used to determine whether biological materials were used to determine whether biological materials were used to determine whether biological materials were used to determine whether biological materials were used to determine whether biological materials were used to determine whether biological materials were used to determine whether biological materials were used to determine whether biological materials were used to determine whether biological materials were used to determine whether biological materials were used to determine whether biological materials were used to determine whether biological materials were Biosensors, sensors including biological materials employed to
  • Biosensor detect and/or monitor an environment, offer several advantages.
  • action potential parameters including action
  • biosensors including live, intact
  • hybrid biosensors have several commercially significant
  • biosensors are particularly useful in detecting
  • Biosensors may also supplement CBW and biological warfare agents.
  • CBW chemical and biological warfare
  • biosensor technology will eliminate, or at least greatly reduce, animal testing
  • cellular membranes are modeled as having a constant capacitance
  • the membrane further, thereby opening more channels, which admit more Na + ,
  • the cell membrane can be made ready in less than a millisecond to
  • Action potential rate refers to the frequency with
  • Action potential which a cell produces an action potential (rapid depolarization).
  • amplitude refers to the height of the peak depolarization that occurs in the course
  • Action potential shape refers to the time course of the
  • the present invention relates to a cell culture chamber 10 having a
  • Figure 1 is a photograph showing the sensor array 16 chip bonded
  • Figures 2 A-C are photomicrographs of the sensor array 16 chip
  • Figure A is the test sensor
  • Figure B is the test sensor
  • FIGS 3 A and B are pictures showing the silicone applied to the
  • Figure A shows a 2 ⁇ m electrode sensor array 16
  • Figure B shows a 2 ⁇ m electrode sensor array 16
  • Figure 4 is a picture showing the image of the entire cell culture
  • Figure 5 is a side view of the preferred embodiment of the cell
  • Figure 6 is another view of the preferred embodiment of the cell
  • Figure 7 is a top view of the preferred embodiment of the cell culture
  • Figure 8 is a photomicrograph at hNT neurons growing on the
  • Figure 9 is a picture of dozens of mammalian cell culture chambers.
  • the present invention provides a cell culture chamber 10
  • sterile environment 12 it is meant that the environment or area
  • the chamber 10 can include a
  • cover 14 it is meant a device sufficient to maintain the sterility of
  • the cover 14 is made of a clear material
  • the cover 14 can also include a semi-permeable membrane. This membrane can be used to cover the cover 14 to cover 14 to cover 14 .
  • This membrane can be used to cover the cover 14 to cover 14 .
  • the present invention permits detection and monitoring of membrane
  • the present invention provides an apparatus
  • Matrigel matrix (Becton Dickinson Labware, Bedford, MA) were used to provide a
  • the sensor array was first coated with poly-D-lysine to promote bonding of
  • the sensor arrays were placed at an incline with
  • Matrigel matrix was thawed overnight in a refrigerator, and then diluted to
  • the sensor arrays could be stored for at least 2 months with one
  • the present invention is useful in screening and assaying
  • Cells are cultured within the medium according to known techniques.
  • a layer of cells (Figure 8) adhere to the respective surfaces of the microelectrode
  • the impedance is determined from this current signal.
  • characteristics include the impedance of the individual cell (i.e., the combined cell
  • the electrodes can also monitor the
  • membrane potential produced by the cell such as neuronal action potentials.
  • action potential parameters include, among others, action potential rate, action
  • each of the microelectrodes is
  • microelectrodes The specific size of the microelectrodes varies according to the
  • the diameter of the cells to be monitored is preferably sized, the diameter of the cells to be monitored.
  • the diameter of the cells to be monitored is preferably sized, the diameter of the cells to be monitored.
  • microelectrodes is less than or equal to one-half the diameter of the cell to
  • microelectrodes with diameters of about 4 to 20 ⁇ m have been utilized.
  • this element can comprise a
  • microelectrode and the reference electrode based on the signals detected with the
  • the signal monitoring and processing means can be any signal monitoring and processing means.
  • the sensors can also be operated to perform amperometric and
  • the microelectrode has a known impedance Z elect . As known in the
  • microelectrode is the resistance of the solution R so , n and the respective
  • Z ref can be determined in a variety of ways. These values, as well as
  • the resistance of the solution, R soln can be factored out, for example, by
  • the cell can be any cell model.
  • the cell can be any cell model.
  • the cell can be any cell model.
  • the cell can be any cell model.
  • the cell can be any cell model.
  • the cell can be any cell model.
  • the cell can be any cell model.
  • the cell can be any cell model.
  • the cell can be any cell model.
  • the cell can be any cell model.
  • the cell can be any cell model.
  • the cell can be
  • models can include a flat, circular "pancake” (i.e., as a disk).
  • Other models can include a
  • synthesizer is used to generate both sine and cosine signals of a programmed
  • the sine voltage signal is attenuated as needed and selectively applied to individual microelectrodes.
  • the resulting signal is detected
  • transimpedance stage which holds the large reference electrode at a
  • control is used to amplify the voltage output of the transimpedance stage.
  • amplified signal is multiplied in quadrature by the source signals and is low pass
  • Quadrature multiplication allows for signal detection, for example, at the excitation
  • a quadrature synthesizer generates both a
  • the generated signals have a frequency of 1 kHz and an amplitude of 10 V peak to
  • the sine wave is attenuated in this example, in a range from 0 to
  • the attenuated signal is then selectively applied to particular
  • microelectrodes In this example, an analog multiplexer is used to apply the
  • the invention is not limited to the number of microelectrodes in the array or to the
  • the resulting current is detected by a transimpedance amplifier
  • the transimpedance amplifier outputs a signal, B sin ( ⁇ + ⁇ ).
  • resistance R sense _ is applied across the input and output of the transimpedance
  • the signal from the transimpedance amplifier is then amplified by an
  • AGC automatic gain control
  • the signal from the AGC amplifier is also mixed with the signal
  • the system is calibrated with a known value
  • phase ⁇ CAL of the calibration values are then determined as follows:
  • ⁇ CAL arc tan [Y CAL /X CAL ]
  • [X CAL 2 +Y CAL 2 ] 1/2
  • ⁇ MEAS arc tan[X MEAS /Y MEAS ]
  • [X MEAS 2 +Y MEAS 2 ] 1/2
  • the detected values for X and Y are sampled with an analog to
  • A/D converter installed in a PC.
  • A/D analog digital
  • processing means such as a PC, receives the detected signal through an (A/D)
  • the output from the AGC amplifier could be A/D converted and input to a
  • the signal monitoring and processing means obtains phase and magnitude
  • processing means provides the capability of performing a spectral analysis to
  • the system can perform spectral analysis of the
  • the membrane which adheres to the respective microelectrodes.
  • the membrane For example, the membrane
  • microelectrodes can be monitored, based on various models of the cells.
  • a test signal oscillator generates sine and cosine signals
  • resulting current is detected using transimpedance amplifier and AGC amplifier.
  • mixers are represented, respectively, by: (AB/2) cos [ ⁇ , F t+ ⁇ ]-(AB/2) cos [(2 ⁇ test + ⁇ IF )t+ ⁇ ](AB/2) sin [ ⁇ IF t+ ⁇ ]-(AB/2) sin [(2 ⁇ test
  • the large reference electrode is then calculated in the manner described above.
  • measurement in the time domain can be any measurement in the time domain.
  • measurement in the time domain can be any measurement in the time domain.
  • each impedance This can be accomplished, for example, by using a step function
  • biocompatible conductive substance such as iridium, activated
  • the substrate of the integrated device may be any organic compound. Additionally, the substrate of the integrated device may be any organic compound. Additionally, the substrate of the integrated device may be any organic compound.
  • glass composed of a variety of materials, such as silicon, glass, metal, quartz, plastic, ceramic, polyethylene, or any other suitable type of polymer. It is noted that glass
  • the passivation layer may comprise any suitable material, including low stress
  • PECVD silicon nitride, silicon carbide, TEFLONTM, polyimide, ceramic, photoresist,
  • Cloning cylinders housed in Petri dishes, are bonded to the
  • the Petri dishes can comprise polystyrene,
  • dishes may be bound to a chip formed in accordance with the invention using
  • thermoplastic using chemical, thermal, or ultrasonic processes.
  • thermal, thermal, or ultrasonic processes In this alternate
  • bondwire connections can be eliminated by use of a substrate
  • a connector such as an edge card connector or
  • substrate area to be significantly larger than the active electrode area.
  • plasma membrane depends on the distribution of ionic charge. Generally, the distribution of ionic charge determines the electric potential, or voltage. For
  • the mobile particles carrying charge are
  • the mobile particles are ions such as Na + , K + ,
  • Nafion acts as a cation exchange membrane (Brazell, et al.,
  • the concentration of the parent bioagent molecule can be
  • gated channels Two classes of gated channels are of
  • channels are not particular to neurons: they are also found in many other types of
  • One goal of the present invention is to augment semiconductor
  • complex molecules with a membrane is to catalize or inhibit a reaction such as the
  • neurotoxins and molecules of biological warfare can be detected by immobilizing antibodies and/or enzymes on the surface of an ion-selective membrane, by
  • organophosphates inhibit the reaction mechanism causing
  • cholinesterase enzymes these can be used to monitor the presence of Paraoxon.
  • the membranes must adhere well to the silicon surface to prevent detachment of
  • membranes include, but are not limited to,
  • enzymes can be immobilized on the membrane surface that
  • organophosphorus compounds such as Paraoxon is to monitor the inhibition of the
  • Membrane adhesion is a critical factor and must be
  • Membranes can be deposited using a set of micropipettes accurate
  • surfaces can print with +/-5 micron alignment and 25 to 50 micron minimum
  • a mammalian cell culture chamber 10 with an integrated silicon
  • the chamber 10 has a
  • cover 14 that allows free transfer of metabolic gases, minimizes medium
  • test stations microscope, experimental setup, laminar flow hood, etc.
  • silicon sensor array 16 is a general purpose device which may be modified to
  • the sensor chips are packaged in a ceramic 100-pin pin grid array
  • the ceramic package body offers
  • the thermal properties of ceramic material provide temperature stability when used for
  • the ceramic material acts as a thermal buffer minimizing temperature
  • Epoxy Patch 1 C (Dexter Corporation)
  • the bonded ceramic packages are heated to
  • Epoxy Patch is carefully applied to the hot packages with
  • a small, sterile cloning cylinder (Fisher Scientific, Chicago, IL) is placed over this
  • the chambers 10 allowed free exchange of metabolic gases
  • the chambers 10 are
  • Keese C.R. et al., "A Whole Cell Biosensor Based on Cell-Substrate Interactions," Annual International Conference of the IEEE Engineering in Medicine and Biology Society, Vol. 12, No. 2, (1990);

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Sustainable Development (AREA)
  • Clinical Laboratory Science (AREA)
  • Molecular Biology (AREA)
  • Cell Biology (AREA)
  • Analytical Chemistry (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analyzing Materials By The Use Of Electric Means (AREA)

Abstract

La présente invention concerne une chambre (10) de culture cellulaire dotée d'un environnement stérile (12) destiné à la culture de cellules, d'un couvercle (14) permettant un transfert de gaz et d'un jeu de détecteurs.
PCT/US2000/041800 1999-11-02 2000-11-02 Chambre de culture de cellules mammaliennes WO2001048144A2 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU55155/01A AU5515501A (en) 1999-11-02 2000-11-02 Mammalian cell culture chamber
US10/129,248 US6852525B1 (en) 1999-11-02 2002-11-02 Mammalian cell culture chamber

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US16347099P 1999-11-02 1999-11-02
US60/163,470 1999-11-02

Publications (2)

Publication Number Publication Date
WO2001048144A2 true WO2001048144A2 (fr) 2001-07-05
WO2001048144A3 WO2001048144A3 (fr) 2001-12-20

Family

ID=22590153

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2000/041800 WO2001048144A2 (fr) 1999-11-02 2000-11-02 Chambre de culture de cellules mammaliennes

Country Status (2)

Country Link
AU (1) AU5515501A (fr)
WO (1) WO2001048144A2 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005168494A (ja) * 2003-11-17 2005-06-30 Hitachi Ltd 細胞培養容器、及び培養細胞
JP2005312343A (ja) * 2004-04-28 2005-11-10 Hitachi Ltd 観察用容器及び培養用容器，培養細胞
EP1541670A4 (fr) * 2002-08-26 2008-07-23 Japan Science & Tech Agency Microchambre destinee a la culture de cellules nerveuses
WO2010148392A1 (fr) 2009-06-19 2010-12-23 University Of Maryland Baltimore County Détection non invasive de paramètres de bioprocédés
WO2011000572A1 (fr) 2009-07-02 2011-01-06 Patenthandel Portfoliofonds I Gmbh & Co. Kg Procédé et dispositif permettant de mettre en évidence des effets biologiques à long terme dans des cellules

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5810725A (en) * 1993-04-16 1998-09-22 Matsushita Electric Industrial Co., Ltd. Planar electrode
US5563067A (en) * 1994-06-13 1996-10-08 Matsushita Electric Industrial Co., Ltd. Cell potential measurement apparatus having a plurality of microelectrodes
US5981268A (en) * 1997-05-30 1999-11-09 Board Of Trustees, Leland Stanford, Jr. University Hybrid biosensors

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1541670A4 (fr) * 2002-08-26 2008-07-23 Japan Science & Tech Agency Microchambre destinee a la culture de cellules nerveuses
JP2005168494A (ja) * 2003-11-17 2005-06-30 Hitachi Ltd 細胞培養容器、及び培養細胞
JP2005312343A (ja) * 2004-04-28 2005-11-10 Hitachi Ltd 観察用容器及び培養用容器，培養細胞
WO2010148392A1 (fr) 2009-06-19 2010-12-23 University Of Maryland Baltimore County Détection non invasive de paramètres de bioprocédés
EP2443224A4 (fr) * 2009-06-19 2014-10-22 Univ Maryland Détection non invasive de paramètres de bioprocédés
WO2011000572A1 (fr) 2009-07-02 2011-01-06 Patenthandel Portfoliofonds I Gmbh & Co. Kg Procédé et dispositif permettant de mettre en évidence des effets biologiques à long terme dans des cellules

Also Published As

Publication number Publication date
AU5515501A (en) 2001-07-09
WO2001048144A3 (fr) 2001-12-20

Similar Documents

Publication Publication Date Title
US6852525B1 (en) Mammalian cell culture chamber
US6051422A (en) Hybrid biosensors
Xu et al. Cell-based biosensors based on light-addressable potentiometric sensors for single cell monitoring
Pancrazio et al. Development and application of cell-based biosensors
CN2372689Y (zh) 电流型生物传感器
Ding et al. Trends in cell-based electrochemical biosensors
Mamouni et al. Interdigitated microelectrode-based microchip for electrical impedance spectroscopic study of oral cancer cells
EP3464551B1 (fr) Détection d'agents infectieux viables dans un échantillon de fluide et sensibilité des agents infectieux à des agents anti-infectieux
US20060160144A1 (en) Sensor arrangement, device and method for testing active substances and/or active sites from a pharmacological point of view using an amperometer and/or potentiometer
US20040040868A1 (en) Microfabricated sensor arrays for multi-component analysis in minute volumes
Bao et al. Recent advances in electric analysis of cells in microfluidic systems
JPH11503316A (ja) 測定装置
EP1230539A1 (fr) Reseau de capteurs microscopiques multisites a circuits d'analyse et de commande integres
Yang et al. Biosensors and their Applications
Mross et al. Integrated multi-sensor system for parallel in-situ monitoring of cell nutrients, metabolites, cell density and pH in biotechnological processes
Thompson et al. Biosensors and bioprobes
Urban et al. Performance of integrated glucose and lactate thin-film microbiosensors for clinical analysers
JP2009544947A (ja) 細胞に関するオンライン測定のための装置
WO2001048144A2 (fr) Chambre de culture de cellules mammaliennes
Wagner et al. Light-addressable potentiometric sensors (LAPS): recent trends and applications
Eiler et al. Application of a thin-film transistor array for cellular-resolution electrophysiology and electrochemistry
Borghol et al. Monitoring of E. coli immobilization on modified gold electrode: A new bacteria-based glucose sensor
US20050227139A1 (en) Device and methods for carring out electrical measurements on membrane bodies
WO2004046707A1 (fr) Reseau microscopique de detecteurs multisites a circuit de commande et d'analyse integre
Primiceri et al. Development of EIS cell chips and their application for cell analysis

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CR CU CZ DE DK DM DZ 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 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: A2

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 GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
AK Designated states

Kind code of ref document: A3

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CR CU CZ DE DK DM DZ 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 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: A3

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 GW ML MR NE SN TD TG

WWE Wipo information: entry into national phase

Ref document number: 10129248

Country of ref document: US

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载