+

WO1999007831A1 - Procede visant l'augmentation ex vivo du nombre des cellules souches hematopoietiques - Google Patents

Procede visant l'augmentation ex vivo du nombre des cellules souches hematopoietiques Download PDF

Info

Publication number
WO1999007831A1
WO1999007831A1 PCT/EP1998/004882 EP9804882W WO9907831A1 WO 1999007831 A1 WO1999007831 A1 WO 1999007831A1 EP 9804882 W EP9804882 W EP 9804882W WO 9907831 A1 WO9907831 A1 WO 9907831A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
stem cells
interleukin
hematopoietic stem
expansion
Prior art date
Application number
PCT/EP1998/004882
Other languages
English (en)
Inventor
Lorenza Lazzari
Paolo Rebulla
Girolamo Sirchia
Original Assignee
Dompe' S.P.A.
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 Dompe' S.P.A. filed Critical Dompe' S.P.A.
Priority to AU88087/98A priority Critical patent/AU8808798A/en
Publication of WO1999007831A1 publication Critical patent/WO1999007831A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/145—Thrombopoietin [TPO]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/26—Flt-3 ligand (CD135L, flk-2 ligand)

Definitions

  • a large number of onco-hematologic diseases can often be treated only with bone-marrow transplantation. It is rather difficult to find a donor with identical HLA within the patient's family and thus, hematopoietic stem cell transplantation from non-related donors represents a novel therapy for those patients who need bone-marrow transplantation but do not have donor- siblings with identical HLA.
  • GVDH graft versus host disease
  • Cord blood hematopoietic stem cells can reconstitute the human hematopoietic system and thus they represent an alternative to bone marrow stem cells.
  • the following advantages are associated with stem cells available from placental blood (stored in cord blood banks as cryopreserved cell collections), in comparison with bone marrows available from donor registries,:
  • CMV cytomegaloviru ⁇
  • Placental blood can be cryopreserved for many years and it is available in short time; 4. a higher possibility to find a donor also for ethnic minorities;
  • cord blood in adult patients has encountered some problems due to the content of stem cells in a cord blood unit, which is frequently not sufficient for transplantation in adults (Kurtzberg J. et al.., N. Eng. J. Med . 335:157-166, 1996; Wagner JE et al.., Lancet 346:214-219, 1995; Locatelli F. et al .. , Bone Marrow Transplant 18:1095-1101, 1996).
  • the ex-vivo expansion is an interesting approach for maintaining hematopoietic stem cells both qualitatively and quantitatively.
  • the cell maturation obtained through the expansion represents a crucial point for the achievement of a suitable clinical application of the product.
  • the expansion effectiveness and the maturation of the expanded cells can be influenced by many factors among which the static or bioreactor culture, the growth factors and the incubation period are the most critical ones (Haylock
  • hematopoietic stem cell self-renewal a variety of culture conditions have been defined which promote expansion of committed progenitor cells, conditions do not presently exist that permit long-term expansion of stem cells (Zijlmans JMJ et al .. Proc Natl Acad Sci USA 92:8901-5, 1995; Zijlmans JMJ et al.. Proc Natl Acad Sci USA 95:725-9, 1998; Bradford GB et al.. Exp Hematol 25:445-53, 1997; Rebel VI et al .. Blood 83: 128-36, 1994).
  • Haylock et al .. achieved a 66-fold expansion using 6 growth factors (IL-1, IL-3, IL-6, G- CSF, SCF) and a 14-day liquid culture.
  • Brugger et al .. obtained a 190-fold expansion with 6 factors (IL-3, IL- 1, IL-6, SCF, EPO, interferon-g ) in 12-14 days.
  • IL-3 and SCF are the most used cytokines for obtaining a precursor expansion, even if the addition of these growth factors has a negative effect, because they induce maturation of primitive stem cells (e.g. towards the neutrophil subpopulation ) at the expense of self-renewal .
  • the CD34+38- immunophenotype defines a primitive ⁇ ubpopulation of progenitor cells and the maintenance of these cells continues to be a subject of ongoing study (randal TD et al .. Blood 87:4057, 1996). Now, a method has been found for the ex-vivo expansion of stem cells which allows to maintain a part of the cell population m form of CD34+ non-committed cells having self-renewal propertie ⁇ . Thi ⁇ method allows to maintain the expansion m constant proliferative growth up to 15 week ⁇ or more, thu ⁇ obtaining the desired qualitative (CD34+/38- phenotype ) and quantitative (cell number) expansion.
  • the method according to the invention comprises culturmg stem cells in a culture medium containing a mixture of cytokmes/growth factors consisting of thrombopoietm (TPO), mterleukm 6, mterleukm 11 (IL- 6 and IL-11) and flt-3 ligand (FL).
  • TPO thrombopoietm
  • IL- 6 and IL-11 mterleukm 11
  • FL flt-3 ligand
  • the culture medium which is a further object of the invention, contains also other conventional components such as fetal bovine serum or albumin.
  • the culture of stem cells which can be obtained, for example, from cord blood or from other sources (e.g. bone-marrow or peripheral blood), is carried out under humidified atmo ⁇ phere containing 5% C0 2 for period ⁇ of time ranging from few days (for example 10 days) to several weeks (20 or more).
  • Figure 1 shows ⁇ the cellular expansion obtained using the method and the medium of the invention, therefore after culture of purified CD34+ cells in the presence of serum-free medium and FL+TPO+IL-6+I -ll .
  • the Cd34+ cells were purified through separation columns and cultured for 15 weeks in serum-free medium and FL+TPO+IL-6+IL-ll . Every week the cultures were demipopulated and cytokines and fresh medium were added. From the data reported in figure 1, a 10 ⁇ -fold expansion of the number of nucleated cells is observed and it is clear that an expansion higher than 10 6 can be obtained just after 15-weeks culture.
  • Figure 2 shows ⁇ the median of the expansion of CD34+/38- cells obtained after culture in the serum free medium containing FL+TPO+IL-6+IL-ll .
  • the cells were stained after expansion and counted by flow cytomet ⁇ c analysis using a FACScan analyzer. This figure show ⁇ that the number of CD34+/CD38- non-committed cells is 100.000 times higher than the initial number.
  • Figure 3 show ⁇ the median of expan ⁇ ion of CFU-GM colonies obtained in pre ⁇ ence of serum-free medium containing FL+TPO+IL-6+IL-ll . The cells were seeded in methylcellulose after expansion. This figure show ⁇ that the number of CFU-GM colonies i ⁇ 100.000 time ⁇ higher than the initial number.
  • Cord Blood sample ⁇ were collected after cesarean ⁇ ection or vaginal delivery.
  • Cord blood wa ⁇ collected by venipuncturing the main vessel at the free end of the cord using a Maco Pharma bag with 29 ml citrate- pho ⁇ phate-dextro ⁇ e (CPD) a ⁇ anticoagulant. The placenta was washed out to collect the blood that remains in vessels.
  • Mononuclear cells were isolated by Ficoll gradient (1.077 g/ml; Lympholite-H, Cedarlane Laboratories, Ontario, Canada) and CD34+ cells were purified through separation columns (CellPro Inc,
  • CD34+ target cell fraction An aliquot of the CD34+ target cell fraction wa ⁇ analyzed to determine purity by flow cytometry.
  • the final recovery of CD34+ cells ranged from 70% to 98% of the initial CD34+ population and the analysis of the enriched cell fraction, performed with an anti-CD34+ monoclonal antibody (Becton Dickinson, Mountain View, California, USA) revealed a purity of 85% to 98% CD34+ cells.
  • the trypan-blue dye exclusion test showed a viability of 96%-99%.
  • the CD34+ cells were seeded for the clonogenic assay. iquid cultures of CD34+ cells CD34+ cells were plated at 3-5xl0 4 /ml, in Tissue Culture Flasks. A serum-free medium containing different concentrations of growth factors a ⁇ indicated wa ⁇ used.
  • TPO TPO, FL, IL-6 and IL-11 (Peprotech EC Ltd, London ,
  • Colony-forming units were evaluated in 35 mm dishe ⁇ by plating 2.5xl0 3 nucleated cells in 1 ml medium containing 0.9% methylcellulose , 30% FBS , 1% BSA, 10 ⁇ 4 M 2-mercaptoethanol , 3 U/ml erythropoietin , 50 ng/ml SCF, 10 ng/ml GM-CSF, 10 ng/ml IL-3 (StemCell Technologies, Vancouver, Canada). After 14 days of culture at 37°C in a 5% CO, fully humidified atmosphere, culture ⁇ larger than 50 cells were scored by microscopy a ⁇ colony forming cells (CFC), i.e.
  • CFC colony forming cells
  • CFU-GM containing granulocytes and macrophage ⁇
  • BFU-E Burst Forming Unit- Erythroid
  • CFU-GEMM containing myeloid cells, erythroid cell ⁇ and egakaryocyte ⁇
  • CD34+ cultured cell ⁇ were ⁇ tained with one or more of the following monoclonal antibodie ⁇ : anti-CD34, -CD61 (gpllla), -CD38.
  • anti-CD34, -CD61 (gpllla), -CD38 For flow cytometry analy ⁇ is 5xl0 5 cells were incubated with monoclonal antibodies for 30 minutes at 4°C and wa ⁇ hed twice in PBS. Cells were analyzed by flow cytometry using a FACScan analyzer (Becton Dickinson) equipped with a filter set for FITC-PE dual- color fluorescence. The percent of stained cells was determined a ⁇ compared to PE- and FITC-conjugated mouse IgGl isotypic control (Becton Dickinson). Cell viability was evaluated by staining cell ⁇ with 7-AAD, and viable cells were gated.
  • the NOD/SCID mice were sublethally irradiated immediately before intravenous tail-vein injection containing an appropriate number of expanded cells. The duration of this assay was 8 weeks.
  • Flow cytometric analysis was performed on peripheral blood and bone marrow after death of mice.
  • the presence of cells positive for human CD34+, CD19+, CD42a+ antigens wa ⁇ relevant after tran ⁇ plantation (until to 15-19%, 14-16%, 13-15% of the total cell ⁇ respectively).
  • All colonie ⁇ obtained after culturing bone marrow cell ⁇ (clonogenic assay), were plucked from plates and were analyzed by a human-specific PCR (Polymerase Chain Reaction): the PCR ⁇ ignal ⁇ were positive for the human Cart-1 gene. Controls consi ⁇ ted of PCRs for human Cart-1 from human peripheral blood leukocytes (positive) and normal mouse bone marrow cell ⁇ (negative). The signals were of human origin.
  • This assay has indicated the capacity of these expanded cells to home the mice bone marrows.

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Hematology (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un procédé visant l'augmentation ex vivo du nombre des cellules souches hématopoïétiques et un milieu de culture destiné aux cellules souches.
PCT/EP1998/004882 1997-08-07 1998-08-05 Procede visant l'augmentation ex vivo du nombre des cellules souches hematopoietiques WO1999007831A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU88087/98A AU8808798A (en) 1997-08-07 1998-08-05 Method for the ex-vivo expansion of hematopoietic stem cells

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ITMI97A001898 1997-08-07
IT97MI001898A IT1293827B1 (it) 1997-08-07 1997-08-07 Metodo per l'espansione ex vivo di cellule staminali emopoietiche

Publications (1)

Publication Number Publication Date
WO1999007831A1 true WO1999007831A1 (fr) 1999-02-18

Family

ID=11377736

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP1998/004882 WO1999007831A1 (fr) 1997-08-07 1998-08-05 Procede visant l'augmentation ex vivo du nombre des cellules souches hematopoietiques

Country Status (3)

Country Link
AU (1) AU8808798A (fr)
IT (1) IT1293827B1 (fr)
WO (1) WO1999007831A1 (fr)

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001032841A3 (fr) * 1999-10-29 2002-02-07 Us Health Methode de differentiation in vitro de lymphocytes t generes a partir de cellules souches cd34?+¿
DE10227611A1 (de) * 2002-06-20 2004-01-15 Bionethos Holding Gmbh Verfahren und Vorrichtung zur Vermehrung und Differenzierung von Zellen in Anwesenheit von Wachstumsfaktoren und einer biologischen Matrix oder Trägerstruktur
WO2003046161A3 (fr) * 2001-11-30 2004-02-12 Universitaetsklinikum Hamburg Procede d'expansion et de differenciation ex vivo de cellules souches multipotentes
EP1453949A4 (fr) * 2001-11-09 2004-12-22 Viacell Inc Production de suspensions cellulaires
US7312078B2 (en) 1998-02-17 2007-12-25 Gamida Cell Ltd. Methods of controlling proliferation and differentiation of stem and progenitor cells
US7344881B2 (en) 2002-01-25 2008-03-18 Gamida Cell Ltd. Methods of expanding stem and progenitor cells and expanded cell populations obtained thereby
US8846393B2 (en) 2005-11-29 2014-09-30 Gamida-Cell Ltd. Methods of improving stem cell homing and engraftment
US9175266B2 (en) 2012-07-23 2015-11-03 Gamida Cell Ltd. Enhancement of natural killer (NK) cell proliferation and activity
US9567569B2 (en) 2012-07-23 2017-02-14 Gamida Cell Ltd. Methods of culturing and expanding mesenchymal stem cells
US10047345B2 (en) 2012-02-13 2018-08-14 Gamida-Cell Ltd. Culturing of mesenchymal stem cells with FGF4 and nicotinamide

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0627487A2 (fr) * 1993-05-24 1994-12-07 Immunex Corporation Liants pour les récepteurs FLT3
WO1997016535A2 (fr) * 1995-10-30 1997-05-09 Novartis Ag PROCEDES D'UTILISATION DE LIGANDS Mpl AVEC DES CELLULES SOUCHES HUMAINES PRIMITIVES

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0627487A2 (fr) * 1993-05-24 1994-12-07 Immunex Corporation Liants pour les récepteurs FLT3
WO1997016535A2 (fr) * 1995-10-30 1997-05-09 Novartis Ag PROCEDES D'UTILISATION DE LIGANDS Mpl AVEC DES CELLULES SOUCHES HUMAINES PRIMITIVES

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
OHMIZONO Y. ET AL: "Thrombopoietin augments ex vivo expansion of human cord blood-derived hematopoietic progenitors in combination with stem cell factor and flt3 ligand", LEUKEMIA, vol. 11, no. 4, April 1997 (1997-04-01), pages 524 - 530, XP002053902 *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7312078B2 (en) 1998-02-17 2007-12-25 Gamida Cell Ltd. Methods of controlling proliferation and differentiation of stem and progenitor cells
US7429489B2 (en) 1998-09-29 2008-09-30 Gamida Cell Ltd. Methods of controlling proliferation and differentiation of stem and progenitor cells
WO2001032841A3 (fr) * 1999-10-29 2002-02-07 Us Health Methode de differentiation in vitro de lymphocytes t generes a partir de cellules souches cd34?+¿
EP1453949A4 (fr) * 2001-11-09 2004-12-22 Viacell Inc Production de suspensions cellulaires
WO2003046161A3 (fr) * 2001-11-30 2004-02-12 Universitaetsklinikum Hamburg Procede d'expansion et de differenciation ex vivo de cellules souches multipotentes
US7344881B2 (en) 2002-01-25 2008-03-18 Gamida Cell Ltd. Methods of expanding stem and progenitor cells and expanded cell populations obtained thereby
DE10227611A1 (de) * 2002-06-20 2004-01-15 Bionethos Holding Gmbh Verfahren und Vorrichtung zur Vermehrung und Differenzierung von Zellen in Anwesenheit von Wachstumsfaktoren und einer biologischen Matrix oder Trägerstruktur
US8846393B2 (en) 2005-11-29 2014-09-30 Gamida-Cell Ltd. Methods of improving stem cell homing and engraftment
US10047345B2 (en) 2012-02-13 2018-08-14 Gamida-Cell Ltd. Culturing of mesenchymal stem cells with FGF4 and nicotinamide
US9175266B2 (en) 2012-07-23 2015-11-03 Gamida Cell Ltd. Enhancement of natural killer (NK) cell proliferation and activity
US9567569B2 (en) 2012-07-23 2017-02-14 Gamida Cell Ltd. Methods of culturing and expanding mesenchymal stem cells

Also Published As

Publication number Publication date
ITMI971898A1 (it) 1999-02-07
AU8808798A (en) 1999-03-01
IT1293827B1 (it) 1999-03-10

Similar Documents

Publication Publication Date Title
EP1753859B1 (fr) Methode de production de globules rouges
EP0812201B1 (fr) Amplification in vitro de cellules souches
Kawada et al. Rapid ex vivo expansion of human umbilical cord hematopoietic progenitors using a novel culture system
Sandstrom et al. Effects of CD34+ cell selection and perfusion on ex vivo expansion of peripheral blood mononuclear cells
Mayani et al. Biology of human hematopoietic stem and progenitor cells present in circulation
Yamaguchi et al. Serum-free coculture system for ex vivo expansion of human cord blood primitive progenitors and SCID mouse-reconstituting cells using human bone marrow primary stromal cells
US20080118477A1 (en) Umbilical cord mesenchymal stem cells support cord blood hematopoiesis
Yamaguchi et al. Umbilical vein endothelial cells are an important source of c‐kit and stem cell factor which regulate the proliferation of haemopoietic progenitor cells
Möbest et al. Serum‐free ex vivo expansion of CD34+ hematopoietic progenitor cells
JP2011501960A (ja) 月経血幹細胞と共に臍帯血由来細胞を共培養する方法
Almici et al. Clonogenic capacity andex vivo expansion potential of umbilical cord blood progenitor cells are not impaired by cryopreservation
WO1999007831A1 (fr) Procede visant l'augmentation ex vivo du nombre des cellules souches hematopoietiques
JP2002543829A (ja) 哺乳動物造血幹細胞のｅｘｖｉｖｏでの増殖
Pick et al. Expansion of megakaryocyte progenitors from human umbilical cord blood using a new two‐step separation procedure
AU2098295A (en) Extracorporeal cell culture and transplantation kits
Fietz et al. Culturing human umbilical cord blood: a comparison of mononuclear vs CD34+ selected cells
CA2219869A1 (fr) Cellule de souche humaine cd-34 hematopoietique
EP1707625A1 (fr) Procede pour produire des cellules souches hematopoietiques ou des cellules precurseurs vasculaires endtheliales
CN119278262A (zh) 方法
US20060084170A1 (en) Cytokine-free growth and maintenance of progenitor cells
Shaw et al. Expansion of megakaryocyte precursors and stem cells from umbilical cord blood CD34+ cells in collagen and liquid culture media
Pierelli et al. Generation of multinuclear tartrate‐resistant acid phosphatase positive osteoclasts in liquid culture of purified human peripheral blood CD34+ progenitors
Miller et al. Ex vivo expansion of human and murine hematopoietic stem cells
Sasayama et al. Expansion of megakaryocyte progenitors from cryopreserved leukocyte concentrates of human placental and umbilical cord blood in short-term liquid culture
KR101149007B1 (ko) 생착능이 증가된 조혈줄기세포의 제조방법

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE GH GM HR HU ID IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW SD SZ 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 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)
NENP Non-entry into the national phase

Ref country code: KR

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA

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