EP2198002A1 - Séparation de populations cellulaires co-cultivées - Google Patents

Séparation de populations cellulaires co-cultivées

Info

Publication number
EP2198002A1
EP2198002A1 EP08804915A EP08804915A EP2198002A1 EP 2198002 A1 EP2198002 A1 EP 2198002A1 EP 08804915 A EP08804915 A EP 08804915A EP 08804915 A EP08804915 A EP 08804915A EP 2198002 A1 EP2198002 A1 EP 2198002A1
Authority
EP
European Patent Office
Prior art keywords
cell populations
cells
populations
cultured
cultured cell
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP08804915A
Other languages
German (de)
English (en)
Inventor
Ute SCHÄFER
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
AVISO MECHATRONIC SYSTEMS GmbH
Original Assignee
AVISO MECHATRONIC SYSTEMS GmbH
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 AVISO MECHATRONIC SYSTEMS GmbH filed Critical AVISO MECHATRONIC SYSTEMS GmbH
Publication of EP2198002A1 publication Critical patent/EP2198002A1/fr
Withdrawn legal-status Critical Current

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
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/04—Cell isolation or sorting

Definitions

  • the invention relates to the use of a device and a method for separating co-cultivated cell populations.
  • Co-cultivation is possible for various biological systems (tissue / cell types).
  • animal and human embryonic stem cells are often cultured primarily on a feeder cell monolayer, the feeder cells providing growth factors.
  • Feeder cells are used, for example, for breeding stem cells.
  • Cells of an embryo are dissociated and cultured as monolayers.
  • the heterogeneous cell population can be inactivated by irradiation or mitomycin treatment, so that the cells can no longer divide.
  • the feeder cells are still metabolically active. Proliferation of embryonic stem cells is dependent on substances released into the medium by feeder cells. To differentiate stem cells, the total culture, including the feeder cells, is removed from the culturing plate used.
  • the stem cells can first develop into so-called hanging drops to form "embryoid bodies.” These "embryoid bodies” are a mixed culture of stem cells and feeder cells. Even in the next differentiation steps, in which the embryoid bodies differ in different cell types by the addition of specific messenger substances. The cultures are contaminated by feeder cells. Since the stem cells divide in the early differentiation phases, but the spring cells were inactivated, the quantitative ratio of the stem cell and feeder cell content changes. In the case of co-cultivation with feeder cells, the stem cells are therefore not pure, but in mixture with feeder cells. Feeder cell contamination can also be demonstrated to a minor extent in differentiated cell populations.
  • the object of the invention is thus to separate co-cultured cell populations.
  • the object is achieved by the use of a device for separating at least two co-cultured cell populations with the features of claim 1 and with a method for separating at least two co-cultured cell populations with the features of claim 5.
  • the subclaims contain expedient or advantageous embodiments and features of the method or the use.
  • a device for the separation of at least two cultured cell populations, which comprises a microscope unit (1) for the microscopic scanning of the cell culture (8), which co-cultured at least two Cell populations, in combination with an image capture unit (2) and an image evaluation unit (3) for detecting the position of cells in the cell culture (8), a control and storage unit (4) for storing the detected position of the cells and a harvesting module (5 ) with a removal tool (10a) for withdrawing the cells at the detected position of the cell.
  • co-cultured cell populations As part of the separation of different co-cultured cell populations, various combinations of cell populations are possible. In particular, at least two co-cultured cell populations may be separated comprising feeder cell and stem cell populations. Since the aspiration conditions can be adapted to the culture conditions, the methodology can also be applied to the separation of other co-cultured cell populations. Other embodiments relate to the use of separating endothelial cell populations and smooth muscle cell populations, as well as separating endothelial cell and liver cell populations.
  • the method according to the invention for separating at least two co-cultured cell populations comprises carrying out a first detection step for selecting cells of a first cell population based on physical and / or physical See parameters and capture position data and store the captured position data of the selected cells in a position database.
  • the method is characterized by the following method steps:
  • Position data from the position database to a harvesting unit is
  • the method corresponds to the method described in international application PCT / EP2007 / 059951. All process specificities disclosed in PCT / EP2007 / 059951 are part of the teaching of the present invention.
  • the method of separating at least two co-cultured cell populations is characterized in that the at least two co-cultured cell populations to be separated comprise feeder cell and stem cell populations. It is also preferred to use the method for the separation of endothelial cell populations and populations of smooth muscle cells and for the separation of endothelial cell and liver cell populations.
  • feeder cells from 13.5-day-old mouse embryos are obtained for the cultivation of stem cells.
  • the embryos are removed organs and the head.
  • the cells of the residual embryo are dissociated and cultured as monolayers.
  • the heterogeneous cell population is treated by irradiation or mitomycin treatment. inactivated so that the cells can no longer divide, but the feeder cells remain metabolically active.
  • the method according to the invention for separating at least two co-cultured cell populations comprises the selection of cells of a first cell population on the basis of physical and / or physical parameters. As can be seen, for example, by light microscopy, feeder cells and stem cells have different structures or shapes (see Figure 1).
  • stem cells grow as almost round colonies on the monolayer of the elongated feeder cells. Accordingly, when using the device of PCT / EP2007 / 059951, surprisingly, the separation of the different cell populations is possible.
  • Stem cells were cultured on neomycin resistant spring cells for 5-8 days. Stem cells of individual colonies were aspirated with the "tool for non-floating cells" under standardized conditions (suction pressure, amount of liquid) .
  • the PCT / EP2007 / 059951 device allowed stem cells to be picked from different colonies within a petri dish
  • the transporter of the feeder cell monolayer with the neomycin resistance gene, this gene is not expressed in the stem cell line D3
  • the separation of stem and feeder cells is shown in Figures 2A to 2E These images show the repeated aspiration of stem cells from a colony (microscopic analysis) Expression analysis (RT-PCR) of the in feeder cells expressed neomycin gene is i Figure 3 can be seen.
  • the microscopic analysis and the subsequent expression analysis illustrate that the aspiration of the stem cells could be repeated several times before the spring cells of the monolayer were aspirated with. Feeder cells were aspirated only after the fifth repetition. Detection was carried out by expression of the neomycin gene in lane E of the electrophoretically separated agarose gel. I 2
  • Embryonic stem cells are pluripotent, ie they can be differentiated into different cell types in vitro.
  • the differentiation potential of feeder-free stem cells and stem cells which were cultivated with feeder cells according to the standard method, based on the neural Differentiation compared.
  • Neuronal differentiation was analyzed by morphological criteria as well as the expression of markers expressed in different phases of neuronal differentiation. At no time were significant differences found in the differentiation potential of feeder-free and stem-cell differentiated stem cells (see Figure 3). It could be shown that a reproducible and complete separation of feeder and stem cells can be achieved and that this method of stem cell purification in no way influences the pluripotency of stem cells (see Figure 4).
  • Fig. 1 Light microscope image of parent
  • Feeder cells stem cells cultured on feeder cells
  • Fig. 2A to 2E repeated aspiration of stem cells of a colony (microscopic analysis)
  • Fig. 3 Expression analysis (RT-PCR) of the in
  • Fig. 4 Detection of the differentiation potential (comparison of feeder-free and according to standard protocol differentiated stem cells)
  • Fig. 5 Apparatus according to PCT / EP2007 / 059951 in an exemplary embodiment

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Sustainable Development (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un procédé et l'utilisation d'un dispositif permettant de séparer des populations cellulaires co-cultivées.
EP08804915A 2007-10-02 2008-09-30 Séparation de populations cellulaires co-cultivées Withdrawn EP2198002A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102007047321A DE102007047321A1 (de) 2007-10-02 2007-10-02 Trennung co-kultivierter Zellpopulationen
PCT/EP2008/063079 WO2009047168A1 (fr) 2007-10-02 2008-09-30 Séparation de populations cellulaires co-cultivées

Publications (1)

Publication Number Publication Date
EP2198002A1 true EP2198002A1 (fr) 2010-06-23

Family

ID=40159441

Family Applications (1)

Application Number Title Priority Date Filing Date
EP08804915A Withdrawn EP2198002A1 (fr) 2007-10-02 2008-09-30 Séparation de populations cellulaires co-cultivées

Country Status (7)

Country Link
US (1) US20100267076A1 (fr)
EP (1) EP2198002A1 (fr)
JP (1) JP2010539963A (fr)
KR (1) KR20100128274A (fr)
CA (1) CA2702016A1 (fr)
DE (1) DE102007047321A1 (fr)
WO (1) WO2009047168A1 (fr)

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7776584B2 (en) * 2003-08-01 2010-08-17 Genetix Limited Animal cell colony picking apparatus and method
WO2008034868A2 (fr) 2006-09-22 2008-03-27 Aviso Gmbh Procédé et dispositif de prélèvement automatique de cellules et/ou de colonies cellulaires

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2009047168A1 *

Also Published As

Publication number Publication date
US20100267076A1 (en) 2010-10-21
WO2009047168A1 (fr) 2009-04-16
JP2010539963A (ja) 2010-12-24
DE102007047321A1 (de) 2009-04-09
CA2702016A1 (fr) 2009-04-16
KR20100128274A (ko) 2010-12-07

Similar Documents

Publication Publication Date Title
EP3159399B1 (fr) Procédé et dispositif de prélèvement automatisé de celles et/ou de colonies de cellules
DE69401991T2 (de) Mehrfachloch Platte für in vito Fertilisation
DE2626733B2 (de) Verfahren zum automatischen und fortgesetzten Züchten lebender Gewebe oder Zellen durch Gewebezucht
EP1910806A1 (fr) Procede et dispositif d'analyse d'objets biologiques
DE202017101988U1 (de) Automatisiertes System zur Kultivierung und Differenzierung pluripotenter Stammzellen
EP2812448A1 (fr) Procédé d'analyse d'acides nucléiques f taux
DE102005021034B4 (de) Verfahren zur Kultivierung einer Zellkultur in einem automatisierten Zellkultursystem sowie automatisiertes Zellkultursystem
WO2009047168A1 (fr) Séparation de populations cellulaires co-cultivées
WO2008034430A2 (fr) Milieu exempt de sérum pour la différenciation de cellules souches
EP1674563A1 (fr) Isolation de cellules souches pluripotentes embryonnaires sans destruction de l'embryon, cellules souches ainsi obtenues et leur utilisation
EP1713899A2 (fr) Procede et dispositif pour la caracterisation de cellules, de groupes cellulaires et/ou de tissus
EP2179029B1 (fr) Procédé et dispositif pour la formation d'agrégats de cellules biologiques
EP3851516A1 (fr) Procédé et dispositif de culture et d'examen de neurones
EP1581613B1 (fr) Procede pour le traitement de cultures cellulaires
WO2015010763A1 (fr) Dispositif et procédé d'encapsulation d'un échantillon dans une capsule polymère
WO2017059946A1 (fr) Injection de globules polaires
Aulich et al. Embryonale Stammzellen
WO2005057179A1 (fr) Element de reception pour recevoir un objet rejete par une masse biologique sous l'effet d'un rayonnement laser
EP2795318B1 (fr) Modèle de peau à gène rapporteur 3d in vitro
Draut The role of retinoic acid in the development of pelvic fins in Danio rerio
DE102008039812A1 (de) Vorrichtung und Verfahren zur Kultivierung von adhärenten Zellen
DE102010043555B4 (de) Verfahren zur Ex-vivo-Kultivierung von Geweben oder embryonalen Organanlagen
Cheng et al. Methods for Studying Axonal Autophagosome Dynamics in Adult Dorsal Root Ganglion Neurons
Neacşu et al. Classification of dorsal root ganglion neurons from newborn rat in organotypic cultures
EP3205729A1 (fr) Procédé destiné à la vérification d'acide nucléique fétal

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20100428

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MT NL NO PL PT RO SE SI SK TR

AX Request for extension of the european patent

Extension state: AL BA MK RS

DAX Request for extension of the european patent (deleted)
RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: AVISO GMBH MECHATRONIC SYSTEMS

19U Interruption of proceedings before grant

Effective date: 20110218

19W Proceedings resumed before grant after interruption of proceedings

Effective date: 20131001

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20140401