ES2539905T3 - Método para aislar y/o identificar células madre mesenquimales (CMM) - Google Patents
Método para aislar y/o identificar células madre mesenquimales (CMM) Download PDFInfo
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- ES2539905T3 ES2539905T3 ES12170880.4T ES12170880T ES2539905T3 ES 2539905 T3 ES2539905 T3 ES 2539905T3 ES 12170880 T ES12170880 T ES 12170880T ES 2539905 T3 ES2539905 T3 ES 2539905T3
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies
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- 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
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2863—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
- C07K16/3015—Breast
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/32—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
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Abstract
Uso de un anticuerpo, o de fragmentos funcionales del mismo, para aislar y/o identificar, de tejido primario, células madre mesenquimales homogéneas, mediante el cual el anticuerpo es el anticuerpo W5C5, que se produce por la línea celular de hibridoma que está depositada, de acuerdo con el Tratado de Budapest, en el Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) con el número DSM ACC 2813.
Description
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E12170880
18-06-2015
Además también existía la posibilidad de diferenciar las células de una manera alternativa, por ejemplo en adipocitos, condrocitos u osteoblastos. En este caso se dio prioridad a las instrucciones relevantes del fabricante (por ejemplo, Miltenyi Biotec). Para diferenciar las CMM en células adipocitarias y osteoblásticas, las células se cultivaron en presencia de medio AdipoDiff NK o medio OsteoDiff NK (ambos de Miltenyi Biotect) siguiendo las instrucciones del fabricante. Para esto, las CMM (12 104 células para la adipogenésis; 4,5 104 células para la osteogénesis) se resuspendieron en 1,5 ml del medio apropiado y se transfirieron a placas de 6 pocillos (Falcon, Heidelberg, Alemania). El medio se renovó cada tres días. La formación de adipocitos se evaluó el día 18 del cultivo fijando las células con metanol durante 5 min. a -20 ºC y tiñendo posteriormente los lípidos intracelulares con tinción de Aceite Rojo O (Sigma-Aldrich) durante 30 min. a temperatura ambiente. La formación de células osteogénicas se analizó el día 10 del cultivo tiñendo la actividad fosfatasa alcalina de las células fijadas con metanol (-20 ºC, 5 min.) con 5-bromo-4-cloro-3-indolil fosfato/nitro azul de tetrazolio (sustrato FAST™ BCPI/NBT; Sigma Aldrich) durante 10 min. a temperatura ambiente.
La Fig. 2 muestra los resultados de los ensayos de UFC-F para las poblaciones separadas. Como puede deducirse de la Fig. 2a (aquí se evaluaron 5000 células), ni las células CD271+CD140b- ni las células CD271+W8B2- tenían actividad de UFC-F significativa. A diferencia de esto, las células CD271+CD140b+, CD271+HEK-3D6+ y CD271+W8B2+ tuvieron actividad de UFC-F fuertemente potenciada en comparación con las células no separadas. Aprox. cada 50-100 células en las fracciones positivas era una CMM con capacidad de UFC-F. De la Fig. 2b (aquí se evaluaron 500 células), puede deducirse que la capacidad de UFC-F es claramente mayor en la fracción CD271+CD56+ que en la fracción CD271+CD56-. En la fracción doble positiva, cada 10 células es una UFC-F.
Como puede deducirse de la Fig. 2, las células CD271 positivas aunque CD140b negativas no mostraron, o solo mostraron, actividad de UFC-F no significativa, al igual que las células CD271 positivas/W8B2-negativas. Por otro lado, las células CD271 positivas/CD140b-positivas o W8B2-positivas mostraron actividad de UFC-F que las diferenciaba como células madre mesenquimales “genuinas”.
La Fig. 3 muestra, por primera vez en la bibliografía, hasta donde son conscientes los autores de la invención, la morfología de CMM separadas de médula ósea. Por consiguiente, las CMM se diferencian por tener una proporción de citoplasma relativamente alta, en la que además parece haber gotas lipídicas. Las imágenes se realizaron con un microscopio Zeiss Axiovert usando el programa informático Axiovision y se evaluaron. Aumento: x 100.
Las Figs. 4a y. 4b muestran imágenes aumentadas de células separadas obtenidas 10 días después de la diferenciación in vitro. En cada caso, en las dos figuras, las células fueron CD271-positivas. En la imagen izquierda, la Fig. 4a muestra células que además se aislaron usando W8B2, y que por consiguiente expresaban el antígeno correspondiente. Como puede observarse en las figuras, las CMM solo pudieron producirse en cultivos de células de la fracción positiva doble (CD271+W8B2+), no de la fracción CD271+W8B2-. La Fig. 4, al igual que la Fig. 4a, muestra un comportamiento de crecimiento análogo para las células de la fracción CD271+CD140b+.
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Claims (1)
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imagen1
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE102006043625 | 2006-09-12 | ||
| DE102006043625A DE102006043625B4 (de) | 2006-09-12 | 2006-09-12 | Verfahren zur Isolierung und/oder Identifizierung von mesenchymalen Stammzellen (MSC) |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| ES2539905T3 true ES2539905T3 (es) | 2015-07-07 |
Family
ID=38926350
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| ES07017866.0T Active ES2539798T3 (es) | 2006-09-12 | 2007-09-12 | Método para aislar y/o identificar células madre mesenquimales (CMM) |
| ES12170880.4T Active ES2539905T3 (es) | 2006-09-12 | 2007-09-12 | Método para aislar y/o identificar células madre mesenquimales (CMM) |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| ES07017866.0T Active ES2539798T3 (es) | 2006-09-12 | 2007-09-12 | Método para aislar y/o identificar células madre mesenquimales (CMM) |
Country Status (4)
| Country | Link |
|---|---|
| US (1) | US8163495B2 (es) |
| EP (2) | EP1901063B1 (es) |
| DE (1) | DE102006043625B4 (es) |
| ES (2) | ES2539798T3 (es) |
Families Citing this family (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| MX2010003019A (es) * | 2007-09-19 | 2010-04-30 | Pluristem Ltd | Celulas adherentes de los tejidos adiposo y de placenta y uso de las mismas en la terapia. |
| DE102008032236A1 (de) | 2008-06-30 | 2010-04-01 | Eberhard-Karls-Universität Tübingen | Isolierung und/oder Identifizierung von Stammzellen mit adipozytärem, chondrozytärem und pankreatischem Differenzierungspotential |
| US10130736B1 (en) | 2010-05-14 | 2018-11-20 | Musculoskeletal Transplant Foundation | Tissue-derived tissuegenic implants, and methods of fabricating and using same |
| US9352003B1 (en) | 2010-05-14 | 2016-05-31 | Musculoskeletal Transplant Foundation | Tissue-derived tissuegenic implants, and methods of fabricating and using same |
| US8883210B1 (en) | 2010-05-14 | 2014-11-11 | Musculoskeletal Transplant Foundation | Tissue-derived tissuegenic implants, and methods of fabricating and using same |
| US8834928B1 (en) | 2011-05-16 | 2014-09-16 | Musculoskeletal Transplant Foundation | Tissue-derived tissugenic implants, and methods of fabricating and using same |
| DE102012102532A1 (de) * | 2012-03-23 | 2013-09-26 | Eberhard-Karls-Universität Tübingen Universitätsklinikum | Neue MSC-Oberflächenmarker |
| US20150037436A1 (en) | 2013-07-30 | 2015-02-05 | Musculoskeletal Transplant Foundation | Acellular soft tissue-derived matrices and methods for preparing same |
| CA2986702C (en) | 2015-05-21 | 2023-04-04 | David Wang | Modified demineralized cortical bone fibers |
| US10912864B2 (en) | 2015-07-24 | 2021-02-09 | Musculoskeletal Transplant Foundation | Acellular soft tissue-derived matrices and methods for preparing same |
| US11052175B2 (en) | 2015-08-19 | 2021-07-06 | Musculoskeletal Transplant Foundation | Cartilage-derived implants and methods of making and using same |
| US11452796B2 (en) | 2017-06-30 | 2022-09-27 | Allosource | Cellular bone grafts, and methods of manufacture and use |
| WO2021144995A1 (ja) * | 2020-01-16 | 2021-07-22 | ＰｕＲＥＣ株式会社 | 高純度間葉系幹細胞 |
| CN112080464B (zh) * | 2020-09-16 | 2022-05-13 | 中国科学院昆明动物研究所 | 一种犬脐带来源的间充质干细胞培养基和培养方法 |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5837539A (en) * | 1990-11-16 | 1998-11-17 | Osiris Therapeutics, Inc. | Monoclonal antibodies for human mesenchymal stem cells |
| US6767541B2 (en) | 2000-03-20 | 2004-07-27 | The Regents Of The University Of California | HER-2/neu overexpression abrogates growth inhibitory pathways |
| EP1365034A3 (en) | 2002-05-21 | 2004-02-18 | Bayer HealthCare AG | Methods and compositions for the prediction, diagnosis, prognosis, prevention and treatment of malignant neoplasia |
| DE10242338A1 (de) | 2002-09-09 | 2004-03-18 | Eberhard-Karls-Universität Tübingen Universitätsklinikum | Antikörper zur Isolierung und/oder Identifizierung mesenchymaler Stammzellen und Verfahren zur Isolierung und/oder Identifizierung mesenchymaler Stammzellen |
-
2006
- 2006-09-12 DE DE102006043625A patent/DE102006043625B4/de not_active Expired - Fee Related
-
2007
- 2007-09-11 US US11/853,604 patent/US8163495B2/en not_active Expired - Fee Related
- 2007-09-12 EP EP07017866.0A patent/EP1901063B1/en not_active Not-in-force
- 2007-09-12 ES ES07017866.0T patent/ES2539798T3/es active Active
- 2007-09-12 EP EP12170880.4A patent/EP2560002B1/en not_active Not-in-force
- 2007-09-12 ES ES12170880.4T patent/ES2539905T3/es active Active
Also Published As
| Publication number | Publication date |
|---|---|
| EP1901063A2 (en) | 2008-03-19 |
| US8163495B2 (en) | 2012-04-24 |
| EP2560002B1 (en) | 2015-04-01 |
| DE102006043625B4 (de) | 2012-02-23 |
| EP2560002A3 (en) | 2013-07-31 |
| US20080075699A1 (en) | 2008-03-27 |
| EP1901063A3 (en) | 2009-11-04 |
| EP1901063B1 (en) | 2015-04-15 |
| ES2539798T3 (es) | 2015-07-06 |
| DE102006043625A1 (de) | 2008-03-27 |
| EP2560002A2 (en) | 2013-02-20 |
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