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• • 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
  • C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
  • C12N5/0629—Keratinocytes; Whole skin
• • 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
  • C12N2503/00—Use of cells in diagnostics
• • 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
  • C12N2503/00—Use of cells in diagnostics
  • C12N2503/04—Screening or testing on artificial tissues
• • 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
  • C12N2503/00—Use of cells in diagnostics
  • C12N2503/04—Screening or testing on artificial tissues
  • C12N2503/06—Screening or testing on artificial skin

Definitions

• the basal layer of the epidermis which has only one cell base, is the germinal compartment. It is at the level of this layer that the proliferation of keratinocytes takes place.
• the basal keratinocytes there is a small proportion of cells called stem cells, which are believed to be responsible for long-term epidermal renewal.
• the immediate offspring of stem cells is called the progenitor population.
• the epidermis thus consists of a heterogeneous set of cells with variable degrees of differentiation (or immaturity). It is generally accepted that basal keratinocytes represent approximately 10% of the whole of the keratinocytes of the epidermis, and the epidermal stem cell compartment only of the order of 0.1%.
• Another object of the present invention is an optimized clonally cultured epithelial cell culture method for evaluating and exploiting specific properties of a single cell, comprising at least the steps of: a) extracting one or more epithelial cells directly from a biological sample of epithelial tissue; b) optionally, selecting at least one population and / or subpopulation of epithelial cells from the cells extracted in step a); c) performing a clonal culture seeded with a distinct and unique epithelial cell directly from step a) or b); and d) qualitatively and / or quantitatively assessing cell growth in the clonal culture of step c).
• the inventors have been able to observe that the growth potential of the cells cultured according to the clonal culture method of the present invention is higher than that of cells cultured according to conventional procedures (see Example B below). .
• the method according to the invention further comprises the step f) of evaluating the tissue reconstruction potential of the cell population of the clonal culture of step c), or of his offspring. More precisely, step f) preferably consists in using the cell population of the clonal culture of step c), or its progeny, to reconstruct a three-dimensional tissue so as to evaluate its tissue reconstruction potential.
• step f) preferably consists in using the cell population of the clonal culture of step c), or its progeny, to reconstruct a three-dimensional tissue so as to evaluate its tissue reconstruction potential.
• cells derived from the primary clonal cultures or micro-cultures can be taken off their culture support and then used individually for each clone of interest to produce a three-dimensional organotypic culture model (for example, an epithelium, epidermis or reconstructed skin).
• the keratinocytes derived from the clonal micro-cultures were removed by trypsination (Gibco) and then placed in mass culture, individually for each clone studied. These cultures were carried out on plastic surfaces on which type I collagen was adsorbed (for example Petri dishes, Biocoat, Becton-Dickinson).
• the culture conditions were equivalent to those used for primary clonal growth: feeder layer of irradiated fibroblasts, culture medium of similar composition. After one week, the cultures reached 50% to 80% confluence.
• the keratinocytes were then removed by trypsination, counted and then reseeded at a density of 2000 to 3000 cells / cm 2 , under identical conditions.
• CFE colony-forming efficiency
• - 1 clone generated a cumulative progeny of 8.48x10 12 keratinocytes in 8 weeks of multiplication in culture (No. 1), which is equivalent to a cumulative average population of 42.95 doublings.
• - 2 clones generated a cumulative progeny of 6.36x10 11 and 2.4x10 11 keratinocytes (No. 2 and No. 3), which equates to 39.21 and 37.80 mean doublings of population, respectively.
• - 1 clone generated cumulative progeny of 2.03x10 10 keratinocytes in 6 weeks of multiplication in culture (No. 4), which is equivalent to a cumulative 34.24 average population doublings.
• - 1 clone generated a cumulative progeny of 2.15x10 9 keratinocytes in 6 weeks of multiplication in culture (No. 5), which equates to a cumulative 31, 00 average population doublings.
• the experiment was carried out at a stage of growth of the clones equivalent to that described in the embodiment example No. 1 (multiplication corresponding to -16 to 17 successive cell generations).
• the technology of parallel clonal micro-cultures makes it possible to precisely estimate the individual clonogenic capacity of the cells of a sample of interest, in this exemplary embodiment a preparation of basal keratinocytes of the epidermis.
• the method is resolutive in defining a clonal growth profile providing a representative functional signature of a tissue, or a sub-localization within a tissue, in this case the basal layer of tissue. the adult human interfollicular epidermis.
• the system makes it possible to distinguish, within a cell sample of interest, cells having distinct potentialities, as a function of their clonal growth capacity in the short term.
• the culture model according to the present invention thus provides an original functional test for estimating the clonogenic capacity of cohorts of cells placed in culture individually.
• One possible application is the development of quality checks carried out at the individual cell scale to evaluate the functionality (or non-functionality) of cellular samples of interest, compared to a validated reference.
• the clonal micro-culture system further provides the ability to generate cell samples from a single cell, each individually corresponding to a precisely defined short-term proliferation capability.
• Another possible application is to use the system for conducting studies to analyze the short-term functional consequences of a treatment (beneficial or toxic) applied at the individual cell level.
• the system makes it possible to demonstrate a genotoxic effect of gamma irradiation on keratinocytes derived from the basal layer of the epidermis.

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• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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• 2008
  • 2008-02-19 FR FR0851054A patent/FR2927633B1/fr not_active Expired - Fee Related
• 2009
  • 2009-02-18 JP JP2010547167A patent/JP2011512146A/ja active Pending
  • 2009-02-18 WO PCT/EP2009/051912 patent/WO2009103731A1/fr active Application Filing
  • 2009-02-18 EP EP09711589A patent/EP2250253A1/de not_active Ceased
  • 2009-02-18 US US12/918,028 patent/US20120264623A2/en not_active Abandoned

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