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

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• Life Sciences & Earth Sciences (AREA)
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• Health & Medical Sciences (AREA)
• Wood Science & Technology (AREA)
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• Biotechnology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Organic Chemistry (AREA)
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• 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)

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• 2007
  • 2007-10-02 DE DE102007047321A patent/DE102007047321A1/de not_active Withdrawn
• 2008
  • 2008-09-30 WO PCT/EP2008/063079 patent/WO2009047168A1/de active Application Filing
  • 2008-09-30 KR KR1020107009771A patent/KR20100128274A/ko not_active Application Discontinuation
  • 2008-09-30 EP EP08804915A patent/EP2198002A1/de not_active Withdrawn
  • 2008-09-30 CA CA2702016A patent/CA2702016A1/en not_active Abandoned
  • 2008-09-30 JP JP2010527430A patent/JP2010539963A/ja not_active Withdrawn
  • 2008-09-30 US US12/680,956 patent/US20100267076A1/en not_active Abandoned

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