PL3064575T3 - Sposób różnicowania pluripotentnych komórek macierzystych indukowanych z mezenchymalnych komórek macierzystych w hepatocyt - Google Patents
Sposób różnicowania pluripotentnych komórek macierzystych indukowanych z mezenchymalnych komórek macierzystych w hepatocytInfo
- Publication number
- PL3064575T3 PL3064575T3 PL13896838T PL13896838T PL3064575T3 PL 3064575 T3 PL3064575 T3 PL 3064575T3 PL 13896838 T PL13896838 T PL 13896838T PL 13896838 T PL13896838 T PL 13896838T PL 3064575 T3 PL3064575 T3 PL 3064575T3
- Authority
- PL
- Poland
- Prior art keywords
- stem cell
- hepatocyte
- differentiating pluripotent
- induced
- mesenchymal stem
- Prior art date
Links
- 210000003494 hepatocyte Anatomy 0.000 title 1
- 210000002901 mesenchymal stem cell Anatomy 0.000 title 1
- 210000001778 pluripotent stem cell Anatomy 0.000 title 1
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
- C12N5/067—Hepatocytes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/37—Digestive system
- A61K35/407—Liver; Hepatocytes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- 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
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
-
- 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
- C12N2500/05—Inorganic components
- C12N2500/10—Metals; Metal chelators
- C12N2500/20—Transition metals
- C12N2500/24—Iron; Fe chelators; Transferrin
- C12N2500/25—Insulin-transferrin; Insulin-transferrin-selenium
-
- 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
- C12N2500/70—Undefined extracts
- C12N2500/76—Undefined extracts from plants
-
- 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
-
- 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
- C12N2501/12—Hepatocyte growth factor [HGF]
-
- 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
- C12N2501/30—Hormones
-
- 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
- C12N2501/30—Hormones
- C12N2501/33—Insulin
-
- 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
- C12N2501/999—Small molecules not provided for elsewhere
-
- 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
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
- C12N2502/1352—Mesenchymal stem cells
- C12N2502/137—Blood-borne mesenchymal stem cells, e.g. Msc from umbilical cord blood
-
- 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
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
-
- 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
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
- C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
- C12N2506/1369—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells from blood-borne mesenchymal stem cells, e.g. MSC from umbilical blood
-
- 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
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Developmental Biology & Embryology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Gastroenterology & Hepatology (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Medicinal Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Hematology (AREA)
- Rheumatology (AREA)
- Nutrition Science (AREA)
- Physiology (AREA)
- Immunology (AREA)
- Virology (AREA)
- Epidemiology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020130132058A KR101542849B1 (ko) | 2013-11-01 | 2013-11-01 | 중간엽 줄기세포로부터 유도된 만능 줄기세포를 이용하여 간세포로 분화시키는 방법 |
| EP13896838.3A EP3064575B1 (en) | 2013-11-01 | 2013-11-05 | Method for differentiating pluripotent stem cell induced from mesenchymal stem cell into hepatocyte |
| PCT/KR2013/009946 WO2015064802A1 (ko) | 2013-11-01 | 2013-11-05 | 중간엽 줄기세포로부터 유도된 만능 줄기세포를 이용하여 간세포로 분화시키는 방법 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| PL3064575T3 true PL3064575T3 (pl) | 2020-08-10 |
Family
ID=53004390
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PL13896838T PL3064575T3 (pl) | 2013-11-01 | 2013-11-05 | Sposób różnicowania pluripotentnych komórek macierzystych indukowanych z mezenchymalnych komórek macierzystych w hepatocyt |
Country Status (18)
| Country | Link |
|---|---|
| US (1) | US10072248B2 (pl) |
| EP (1) | EP3064575B1 (pl) |
| JP (1) | JP6711757B2 (pl) |
| KR (1) | KR101542849B1 (pl) |
| CN (1) | CN105683359B (pl) |
| AU (1) | AU2013403885B2 (pl) |
| CY (1) | CY1122791T1 (pl) |
| DK (1) | DK3064575T3 (pl) |
| ES (1) | ES2776183T3 (pl) |
| HR (1) | HRP20200474T1 (pl) |
| HU (1) | HUE048804T2 (pl) |
| LT (1) | LT3064575T (pl) |
| PL (1) | PL3064575T3 (pl) |
| PT (1) | PT3064575T (pl) |
| RS (1) | RS60363B1 (pl) |
| SI (1) | SI3064575T1 (pl) |
| SM (1) | SMT202000129T1 (pl) |
| WO (1) | WO2015064802A1 (pl) |
Families Citing this family (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| LT3064572T (lt) | 2013-11-01 | 2020-07-27 | Bbhc Co. Ltd. | Indukuotų pluripotentinių kamieninių ląstelių gavimo būdas iš mezenchiminių kamieninių ląstelių ir indukuotos pluripotentinės kamieninės ląstelės, gautos taikant šį metodą |
| KR101699761B1 (ko) * | 2014-05-23 | 2017-01-25 | 주식회사 비비에이치씨 | 플로로탄닌 분획물을 이용한 중간엽 줄기세포로부터 유도만능 줄기세포를 제조하는 방법 |
| KR102018783B1 (ko) * | 2015-08-13 | 2019-09-06 | (주) 넥셀 | 간세포의 약물대사 기능 향상방법 |
| CN105385651B (zh) * | 2015-12-11 | 2019-06-14 | 湖南光琇高新生命科技有限公司 | 诱导性多能干细胞定向诱导分化为肝细胞的方法及肝细胞 |
| WO2018093233A1 (ko) | 2016-11-21 | 2018-05-24 | 성균관대학교산학협력단 | 지방줄기세포유래 엑소좀을 유효성분으로 포함하는 간 섬유증 예방 또는 치료용 조성물 |
| KR20180057546A (ko) | 2016-11-21 | 2018-05-30 | 성균관대학교산학협력단 | 지방줄기세포유래 엑소좀을 유효성분으로 포함하는 간 섬유증 예방 또는 치료용 조성물 |
| WO2020013719A1 (en) * | 2018-07-10 | 2020-01-16 | Uniwersytet Jagielloński | Stem cell culture method using plant secretion |
| CN113194926A (zh) | 2018-12-14 | 2021-07-30 | 图特科技有限公司 | 用于预防或治疗肝病的包括含有间充质干细胞的生物植入物的药物组合物 |
| MX2022011245A (es) | 2020-03-11 | 2023-01-11 | Bit Bio Ltd | Método de generación de células hepáticas. |
| USD1055993S1 (en) | 2023-01-02 | 2024-12-31 | Samsung Electronics Co., Ltd. | Refrigerator |
| USD1092573S1 (en) | 2023-10-16 | 2025-09-09 | Samsung Electronics Co., Ltd. | Refrigerator |
Family Cites Families (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100135970A1 (en) | 2006-10-27 | 2010-06-03 | Caritas St. Elizabeth Medical Center Of Boston, In | Methods for Reprogramming Adult Somatic Cells and Uses Thereof |
| KR20100001940A (ko) | 2008-06-28 | 2010-01-06 | 최송화 | 마우스 내장용 키보드 |
| US20110195056A1 (en) | 2008-07-31 | 2011-08-11 | The General Hospital Corporation | Compositions comprising hepatocyte-like cells and uses thereof |
| KR101016761B1 (ko) | 2009-02-26 | 2011-02-25 | 부경대학교 산학협력단 | 피부 미백활성을 갖는 감태 추출물 |
| KR101073523B1 (ko) | 2009-03-10 | 2011-10-17 | (주)나우이앤씨 | 비개착식 터널 굴착 방법 및 이에 사용되는 터널시공구조체 |
| JP2012533310A (ja) * | 2009-07-23 | 2012-12-27 | 北京華源博創科技有限公司 | 分化誘導による肝細胞、肝内胚葉細胞及び肝前駆細胞を得る方法 |
| JP2012210154A (ja) * | 2009-08-03 | 2012-11-01 | Keio Gijuku | 分化細胞由来多能性幹細胞の樹立方法 |
| WO2011037301A1 (ko) * | 2009-09-22 | 2011-03-31 | 서울대학교병원 | 성체세포로부터 만능줄기세포를 유도하는 방법 및 그 방법에 의해 제조된 만능줄기세포 |
| KR20110079436A (ko) | 2010-03-29 | 2011-07-07 | (주) 세이텍 | 오토틸트형 휴대폰 슬라이딩 힌지 |
| KR20120040488A (ko) | 2010-10-19 | 2012-04-27 | 충남대학교산학협력단 | 곰피와 감태 추출물 유래의 플로로탄닌을 포함하는 화장품 조성물 |
| KR101887956B1 (ko) | 2011-08-10 | 2018-08-13 | (주)아모레퍼시픽 | 효소 처리 감태 추출물을 포함하는 피부 미백용 조성물 |
| CN102286532B (zh) * | 2011-09-05 | 2012-10-17 | 浙江大学 | 一种获得诱导性多能干细胞的方法 |
| KR101408106B1 (ko) | 2011-12-30 | 2014-06-19 | 박영준 | 줄기세포 분화능 개선용 조성물 |
-
2013
- 2013-11-01 KR KR1020130132058A patent/KR101542849B1/ko active Active
- 2013-11-05 HR HRP20200474TT patent/HRP20200474T1/hr unknown
- 2013-11-05 CN CN201380080667.0A patent/CN105683359B/zh active Active
- 2013-11-05 EP EP13896838.3A patent/EP3064575B1/en active Active
- 2013-11-05 DK DK13896838.3T patent/DK3064575T3/da active
- 2013-11-05 RS RS20200314A patent/RS60363B1/sr unknown
- 2013-11-05 LT LTEP13896838.3T patent/LT3064575T/lt unknown
- 2013-11-05 PL PL13896838T patent/PL3064575T3/pl unknown
- 2013-11-05 HU HUE13896838A patent/HUE048804T2/hu unknown
- 2013-11-05 WO PCT/KR2013/009946 patent/WO2015064802A1/ko not_active Ceased
- 2013-11-05 PT PT138968383T patent/PT3064575T/pt unknown
- 2013-11-05 SI SI201331690T patent/SI3064575T1/sl unknown
- 2013-11-05 JP JP2016552367A patent/JP6711757B2/ja active Active
- 2013-11-05 ES ES13896838T patent/ES2776183T3/es active Active
- 2013-11-05 US US15/033,405 patent/US10072248B2/en active Active
- 2013-11-05 SM SM20200129T patent/SMT202000129T1/it unknown
- 2013-11-05 AU AU2013403885A patent/AU2013403885B2/en active Active
-
2020
- 2020-03-16 CY CY20201100237T patent/CY1122791T1/el unknown
Also Published As
| Publication number | Publication date |
|---|---|
| JP2016537022A (ja) | 2016-12-01 |
| DK3064575T3 (da) | 2020-03-16 |
| EP3064575B1 (en) | 2020-02-26 |
| KR101542849B1 (ko) | 2015-08-10 |
| PT3064575T (pt) | 2020-04-02 |
| HRP20200474T1 (hr) | 2020-10-02 |
| RS60363B1 (sr) | 2020-07-31 |
| CN105683359A (zh) | 2016-06-15 |
| AU2013403885A1 (en) | 2016-06-23 |
| EP3064575A1 (en) | 2016-09-07 |
| SMT202000129T1 (it) | 2020-05-08 |
| CY1122791T1 (el) | 2021-05-05 |
| US20160272936A1 (en) | 2016-09-22 |
| SI3064575T1 (sl) | 2020-08-31 |
| WO2015064802A1 (ko) | 2015-05-07 |
| JP6711757B2 (ja) | 2020-06-17 |
| EP3064575A4 (en) | 2017-07-26 |
| ES2776183T3 (es) | 2020-07-29 |
| AU2013403885B2 (en) | 2018-04-05 |
| KR20150050864A (ko) | 2015-05-11 |
| HUE048804T2 (hu) | 2020-09-28 |
| US10072248B2 (en) | 2018-09-11 |
| LT3064575T (lt) | 2020-06-10 |
| CN105683359B (zh) | 2021-08-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| SMT202000129T1 (it) | Metodo di differenziazione di cellule staminali pluripotenti indotte da cellule staminali mesenchimali in epatociti | |
| IL250340A0 (en) | Growth media for the culture of pluripotent stem cells | |
| PT2893001T (pt) | Método para cultura de células estaminais mesenquimatosas | |
| PL3064573T3 (pl) | Sposób różnicowania pluripotentnych komórek macierzystych indukowanych z mezenchymalnych komórek macierzystych w neuron | |
| SMT202000096T1 (it) | Metodo di differenziazione di cellule staminali pluripotenti indotte da cellule staminali mesenchimali in condrociti | |
| EP2949746A4 (en) | METHOD OF SUB-CULTURE OF PLURIPOTENT STEM CELLS | |
| IL249170A0 (en) | A method for generating induced pluripotent stem cells from mesenchymal stem cells using phlorotannin fractionation | |
| ZA201504388B (en) | Method for culturing pluripotent stem cells | |
| EP3064570A4 (en) | Method for differentiating pluripotent stem cell induced from mesenchymal stem cell into adipocyte | |
| PL3156481T3 (pl) | Sposób wytwarzania indukowanych pluripotencjalnych komórek macierzystych z mezenchymalnych komórek macierzystych pochodzących z tkanki tłuszczowej oraz indukowane pluripotencjalne komórki macierzyste wytwarzane tym sposobem | |
| SMT202000121T1 (it) | Metodo di differenziazione di cellule staminali pluripotenti indotte da cellule staminali mesenchimali in osteoblasti | |
| SG11201508796SA (en) | Method for producing induced pluripotent stem cell from mesenchymal stem cell and induced pluripotent stem cell produced by the method |