WO2009144574A1 - Tissus modifiés - Google Patents

Tissus modifiés Download PDF

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Publication number
WO2009144574A1
WO2009144574A1 PCT/IB2009/005778 IB2009005778W WO2009144574A1 WO 2009144574 A1 WO2009144574 A1 WO 2009144574A1 IB 2009005778 W IB2009005778 W IB 2009005778W WO 2009144574 A1 WO2009144574 A1 WO 2009144574A1
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Prior art keywords
cells
tissue
exogenous
modified
colonies
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PCT/IB2009/005778
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English (en)
Inventor
Abdoelwaheb El Ghalbzouri
Cees P. Tensen
Original Assignee
Academisch Zeikenhuis Leiden Acting Under The Name Leiden University Medical Center
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Publication of WO2009144574A1 publication Critical patent/WO2009144574A1/fr

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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
    • C12N2503/04—Screening or testing on artificial tissues
    • C12N2503/06—Screening or testing on artificial 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
    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention concerns the generation of tissues comprising one or more colonies of modified cells, tissues generated by these methods and uses of the same in methods for screening, testing and/or identifying compounds and their effects on a variety of aspects of cell function.
  • the present invention provides a method permitting, for example, the functional screening of hundreds of individual genes in physiologically relevant (human tissue context) systems.
  • the present invention combines methods which permit the introduction of exogenous components into cells with cell and/or tissue culture methods to provide fully differentiated tissues that may be maintained in culture for several weeks and which comprise regions (or colonies) of cells which have been modified to include the exogenous component.
  • the tissues generated by the methods described herein represent physiologically relevant systems in which, for example, the effect of genes, compounds, combinations of compounds and/or varying concentrations, amounts or levels of compounds on, for example, cell morphology, cell function, growth, proliferation, metabolism, differentiation and/or gene expression may be determined.
  • the present invention provides a method of generating a tissue comprising one or more colonies of modified cells, said method comprising the step of introducing into one or more cells an exogenous component and maintaining the cells under conditions suitable to promote the growth, proliferation and/or differentiation thereof, to form a tissue comprising colonies of cells modified to include the exogenous component. It is important to note that of the entire population of cells maintained under the conditions suitable to promote the growth, proliferation and/or differentiation thereof, only those into which the exogenous component has been introduced are to be regarded as having been modified. Accordingly, those cells into which no exogenous component is introduced are to be regarded as "un-modified".
  • the cells are maintained under suitable conditions (described below), they will grow to generate a tissue comprising both modified and un-modified cells, characterised in that the modified cells (i.e. those into which an exogenous component has been introduced) may grow to form one or more discrete colonies within the tissue.
  • discrete is meant that the colonies of modified cells do not touch or overlap and are separated by regions of cells which are un-modified.
  • Conditions suitable to promote the growth, proliferation and/or differentiation of the cells may include the use of growth or culture media and one of skill will readily understand that the present invention may require the use of media suitable for supporting the growth of cells and in particular, the growth of mammalian cells. The skilled man will also understand that a number of media may be suitable for this purpose and as such, for brevity, only a limited number are mentioned herein.
  • Media for use in the present invention may be supplemented with one or more factors to promote the growth, proliferation and/or differentiation of one or more cell types.
  • a particular medium may be supplemented with one or more factors which improve its ability to support the growth of a particular cell, to eliminate the growth of other types of cell and/or to selectively enhance, enrich or promote the growth, proliferation and/or differentiation of a particular cell type.
  • Useful supplementary factors may include cytokines, growth factors, vitamins, antibiotics (antifungals, antivirals and/or antibacterials) and/or mammalian serum (for example bovine foetal serum).
  • a medium for use in any of the methods described here may be supplemented with factors such as foetal calf serum, L- Glutamine, penicillin, streptomycin and mercaptoethanol.
  • Dulbecco's modified Eagle medium DMEM
  • IMDM Iscove's Modified Dulbecco's Medium
  • Ames Medium, Basal Media Eagle, Click's Medium Dulbecco's Modified Eagle's Medium/Ham's Nutrient Mixture F- 12, Dulbecco's Phosphate Buffered Saline, Earle's Balanced Salts, Glasgow Minimum Essential Media, Grace's Insect Media, Hanks' Balanced Salts, IPL-41 Insect Medium, L- 15 Media, M2 and M16 Media, McCoy's 5
  • a Modified Media MCDB Media, Medium 199, Minimum Essential Medium Eagle (MEM), NCTC Media, Nutrient Mixtures (HAM) F-IO, Nutrient Mixtures (HAM) F-12, RPMI-1640 Media, Schneider's Insect Media, Shields and Sang M3 Insect Media, TC-100 Insec
  • condition may also include the use of particular environmental factors such as temperature, radiation (for example UV or gamma radiation), pressure and/or atmospheric composition (for example the use of a low or high oxygen and/or CO 2 concentration). Additionally, or alternatively, the term “conditions” may include the use of particular culture techniques such as liquid or solid media culture or culture at the gas-liquid interface.
  • environmental factors such as temperature, radiation (for example UV or gamma radiation), pressure and/or atmospheric composition (for example the use of a low or high oxygen and/or CO 2 concentration).
  • conditions may include the use of particular culture techniques such as liquid or solid media culture or culture at the gas-liquid interface.
  • tissue may be taken to represent any population cells generated by, for example, those techniques and methods routinely employed in cell/tissue culture.
  • tissue should be taken to refer to a multi-layered cell-mass - i.e. a cell mass comprising at least two layers of cells, and may be taken to mean a collection of cells which work together.
  • a tissue may be considered as distinct ' from a monolayer or "lawn" of cells.
  • the tissue may take the form of a collection of tissues, an organised structure or organ. More specifically, a tissue may comprise numerous layers or populations of one or more cell types. Additionally, or alternatively, a tissue may comprise cells derived from a single cell type but which have been induced to differentiate along distinct pathways such that the resulting tissue comprises differently differentiated cells. More organised structures and organs may comprise one or more tissues which in turn comprise one or more cell types. In certain embodiments, an organised structure or organ may represent the whole or part of an organ naturally present in the body of an organism.
  • the methods described herein may utilise cells derived from the skin, mucosal membranes (including those lining the respiratory, uro/genital and/or intestinal systems) muscle, organs of the circulatory system (including the heart and/or lungs), the kidney, comea, genitalia, reproductive systems, lung and/or neural tissue (including the brain and/or spinal cord).
  • the invention may require the use of cells derived from mammalian skin, such as, for example, normal human keratinocytes.
  • mammalian skin such as, for example, normal human keratinocytes.
  • Suitable sources of keratinocytes may include, for example, human skin tissue samples such as breast skin, foreskin, abdominal skin or the like.
  • the methods of the present invention may utilise cells derived from a variety of organisms, particularly cells of mammalian and/or insect origin. The methods should also be understood as potentially requiring the use of cells derived from organisms at any stage of development and the methods may exploit the use of embryonic, foetal, neonatal, infant and/or adult cells. Additionally, or alternatively, the present invention may require the use of cells derived from samples which comprise one or more parts, portions, biopsies, explants and/or microexplants of a tissue or organ derived from an insect or mammal.
  • Cells derived from mammals such as, for example rodents (mouse, rat, rabbit, hamster or guinea pig) or cells of human origin will be particularly useful.
  • embryonic and/or foetal derived cells and/or tissues material derived from the early, mid and late gestational stages is to be considered as potentially useful in the methods described herein and that material derived from the umbilical cord, embryos and/or foetuses (especially human embryos and/or foetuses) which have been aborted or artificially created, for example as part of a fertility treatment program, are to be considered as suitable sources.
  • the methods described herein may use samples derived from established cell lines (including established adult and/or embryonic stem cell lines), especially those representative of particular tissues, structures or organs.
  • the methods of this invention may utilise cells and biopsies etc., derived from diseased tissue, animal models of disease (in particular the tissues which exhibit symptoms or characteristics of the modelled disease) and cells - possibly derived from cell lines, which may have been isolated from a diseased tissue and which exhibit a morphology, phenotype/geneotype and/or other characteristic typical of a disease or condition.
  • tissue generated by the methods provided by this invention may represent human skin and in particular the epidermis.
  • colony may be taken to mean populations of neighbouring cells and a colony of cells may result from the maintenance of a cell or cells under conditions suitable to promote the growth, proliferation and/or differentiation thereof.
  • the formation of a colony may begin with the growth and/or proliferation of one or more cells - referred to hereinafter as the "seed" cell or cells, resulting in the formation of a population of cells each of which is derived from the seed cell or cells.
  • the seed cell or cells may be genetically identical to the seed cell or cells.
  • the inventors have observed that when a cell is modified to include an exogenous component, that cell will, as a result of division and subsequent differentiation mainly migrate in the vertical direction (particularly after a monolayer has been established). In this way, it is possible to generate a tissue comprising one or more discrete colonies of modified cells which are each surrounded by unmodified cells.
  • exogenous components may be introduced into cells and the particular choice of method may depend upon the nature of the exogenous component to be introduced.
  • component should be understood to include any material which can be introduced into a cell and an "exogenous component” may be taken to be any material which is foreign to or different in origin from, the cell into which it is intended to be introduced.
  • Suitable "exogenous components” for use in the methods described herein may include, for example small molecules, peptides, small organic molecules, antibodies, nucleic acids and/or fragments of any of these.
  • component may also be taken to include organisms which are capable of being introduced into a cell, and may include, for example, viral particles, bacteria, protozoa and the like.
  • the term "compound” may also embrace agents which are capable of being introduced into cells using transfection protocols.
  • transfection protocols utilise conditions which render cell membranes permeable to compounds such as nucleic acids.
  • electroporation heat shock and/or compounds such as calcium phosphate.
  • transfection protocols are used to transfer or introduce nucleic acid (e.g. "foreign" nucleic acid - not derived from the cell into which it is to be introduced) into cells.
  • compound may also encompass agents which are capable of delivering themselves and/or an agent, moiety, compound or substance bound, fused, integrated, linked or otherwise conjugated thereto, to the inside of a cell.
  • agents may include, nucleic acid and/or vectors (for example eukaryotic gene expression vectors) such as transcription cassettes, plasmids and/or bacterial/viral vectors.
  • nucleic acid may include sense or anti-sense DNA or RNA molecules of any length.
  • nucleic acid for use as exogenous component in the methods described herein may be capable of modulating (i.e. up or down-regulating) the expression of one or more genes in a cell.
  • exogenous components for use in the present invention may include inhibitory RNA molecules such as siRNA and/or sh-RNA and or anti-sense oligonucleotides comprising DNA and one of skill in the art will appreciate that algorithms such as BIOPREDs/ can be used to computationally predict nucleic acid sequences that have an optimal knockdown effect for a given gene (see for example: http://www.biopredsi.orgAstart.hlml).
  • nucleic acids useful in the present invention may take the form of whole or partial genes such as, for example disease associated or causing genes.
  • the nucleic acid may be expressed when introduced into a cell.
  • an exogenous component may comprise a nucleic acid sequence encoding one or more genes (for example disease associated genes) and a vector capable of directing the expression of said one or more genes when in a cell.
  • the vector may take the form of an expression vector such as, for example, a viral expression vector or other construct.
  • Vectors suitable for use in the methods described herein and in particular those suitable for use in transfection protocols for delivering exogenous components into cells are known to the skilled man and may include those that are artificially created or those which occur naturally in other organisms.
  • a vector may take the form of a linear of circular nucleic acid molecule.
  • the material (exogenous component) to be introduced into the cell may be contained within, for example, a viral vector.
  • the vector is a lentiviral vector.
  • a vector for use in the methods described herein may be modified so as to comprise a nucleic acid (either a sense or antisense (i.e. inhibitory) DNA or RNA) capable of up- or down-regulating the expression of one or more genes of the cell or cells into which it is to be introduced.
  • a nucleic acid either a sense or antisense (i.e. inhibitory) DNA or RNA
  • the exogenous component may be introduced into a cell by maintaining cells in the presence of an immobilised exogenous component.
  • immobilised it is meant that the component to be introduced into a cell is bound, linked, conjugate, fixed or otherwise coupled to some form of surface or substrate.
  • Suitable surfaces or substrates may include those comprising, for example, polycarbonate, polyester, polystyrene, polyethylene terephthalate membrane, paper, plastic, nitrocellulose, glass, glass fibre, nylon and cellophane membranes.
  • Other suitable substrates may include those comprising fibroblasts that have been embedded in different matricies such as fibrin, matrigel or collagen. These matricies may originate from any one of the sources specified above (for example human, animal or insects).
  • cell derived matricies may include those generated by cells such as fibroblasts onto which other cells such, as, for example, keratinocytes may be seeded.
  • the production of collagen may be accelerated by using genetically modified fibroblasts and/or by manipulation of the media.
  • the choice of surface or substrate upon which the exogenous component is to be immobilised might depend upon the particular form of the exogenous component.
  • immobilise agents such as nucleic acids and vectors, particularly viral vectors
  • substrates such as nitrocellulose.
  • the suitable surface or substrate is permeable such that, for example, a culture media may permeate through.
  • the exogenous component may be formulated as a solution and a small amount deposited or spotted (by, for example, pipette) on to the surface of the chosen surface or substrate and allowed to dry and, depending upon the nature of the substrate and/or exogenous component, the interactions that form between the exogenous component and the surface or substrate may be sufficient to immobilise the exogenous component.
  • the exogenous components and/or the surface/substrate may be necessary to subject the exogenous components and/or the surface/substrate, to some form of protocol which facilitates the immobilisation of the exogenous component. Additionally, or alternatively, it may be necessary to coat the surface or substrate with a substance and/or compound that facilitates the immobilisation of the exogenous component.
  • a substance and/or compound that facilitates the immobilisation of the exogenous component it may be desirable to coat the surface or substrate with, for example, a source of fibronectin.
  • the source of fibronectin may be a recombinant source. Suitable forms of recombinant fibronectin include RetroNectin ® .
  • surfaces might be coated with synthetic peptides/proteins.
  • the substrate comprises cells (e.g. in a scaffold) these cells may be manipulated to produce endogenous proteins for facilitation of transfection and/or immobilisation of exogenous components such as those detailed above.
  • exogenous components described herein may be immobilised onto any suitable surface or substrate either in a single location or in multiple (discrete) locations as an array.
  • more than one exogenous component may be immobilised at any one location and, when arranged or immobilised as an array, it may be desirable to immobilise a number of different exogenous compounds at different locations in the array. In other embodiments the amount, concentration or level of any given exogenous component immobilised at each location may vary.
  • Cells may be "maintained in the presence" of an immobilised exogenous component using a number of techniques. For example, once the exogenous component(s) have been immobilised onto a suitable surface or substrate, the surface or substrate may be contacted with a suspension/solid culture of cells. In one embodiment, the substrate or surface upon which the exogneous component(s) is/are immobilised may be seeded with one or more cells such that those cells attach or adhere to said surface or substrate. Cells which have attached or adhered may, under suitable conditions, continue to grow, proliferate and/or differentiate such that a tissue according to the present invention, may develop. For example, a monolayer of cells may first establish adjacent the immobilised exogenous components.
  • those cells which are adjacent or proximal to an immobilised exogenous component will be modified thereby and, under suitable conditions will grow to form a colony of modified cells.
  • the cell-transfecting agents have been immobilised as an array, certain areas of the surface/substrate may, initially, be devoid of cells - however, provided the cells are maintained under conditions suitable to promote the growth, proliferation and/or differentiation thereof, the cells may eventually cover all areas of the substrate/surface.
  • the present invention is characterised in that the tissue generated by any of the methods described herein may comprise one or more colonies of modified cells and, where there are a plurality of modified cell colonies, each colony is distinct i.e. they are separated by regions of un-modified cells - in other words, the tissue may be considered as comprising an array of modified cell colonies.
  • the duration of the period of maintenance and/or the distance between the immobilised exogenous components may have to be adjusted. The distance between regions of immobilised exogenous component may depend upon the type of cell being cultured and/or the nature of the exogenous component.
  • the cells are known to be highly receptive to transfection and/or where the cells readily uptake exogenous component or where the exogenous component is particularly efficient at entering or transfecting cells, it may be necessary to construct an array of immobilised exogenous component in which the distance between locations (or regions) of immobilised exogenous component is greater than if the cells are poorly receptive to the uptake or transfection of such components and/or where the exogenous component exhibits a low transfection efficiency.
  • cells which grow, proliferate and/or differentiate quickly under certain conditions may require periods of maintenance which are shorter than those periods used when maintaining cells which grow, proliferate and differentiate less quickly.
  • the present invention provides a method of generating epidermis comprising one or more colonies of modified cells, said method comprising the step of introducing into one or more epidermal cells an exogenous component and maintaining the epidermal cells under conditions suitable to promote the growth, proliferation and/or differentiation thereof, to form an epidermis comprising colonies of cells modified to include the exogenous compound.
  • the epidermal cells are keratinocytes and melanocytes derived from normal human tissue (epidermis).
  • the exogenous components may be viral vectors (more preferably lentiviral vectors) which may further comprise elements capable of modulating the expression of one or more genes of the keratinocytes and melanocytes.
  • tissue comprising one or more colonies of cells modified to include an exogenous component.
  • the tissue comprises an array of distinct colonies each of which comprises cells which have been modified to include an exogenous component.
  • said colonies are distinct from one another.
  • tissue obtainable by any of the methods described herein.
  • the fourth aspect of this invention provides a method of screening, testing and/or identifying the effect of a compound in a tissue, said method comprising the steps of
  • test agent introducing into one or more cells, a test agent; (ii) maintaining said cells under conditions suitable to promote the growth, differentiation and/or proliferation thereof such that they form a tissue comprising one or more colonies of cells modified to include the test agent;
  • the test agents may be compounds capable of modulating the expression of one or more genes in a cell. Additionally, or alternatively, the test agents may be compounds which are thought to be potentially useful in the treatment of a disease and/or condition.
  • the test agents may be derived from a library of compounds and as such may take the form of nucleic acid sequences (either DNA or RNA), proteins, peptides/amino acid sequences, small organic molecules, antibodies and/or fragments, portions or homologues of any of these.
  • test agents may be immobilised on to any of the suitable surfaces or substrates described above as an array and one of skill in the art will understand that one or more different test agents may be immobilised at one or more different locations in the array. Furthermore, any given test agent may be immobilised at different concentrations, levels or amounts.
  • the present invention provides methods in which a large number of test agents may be tested, identified and or screened for their effects upon tissues/organs and/or the cells which comprise the tissue. Accordingly, the present invention may provide methods which allow for the high-throughput and/or microarray screening of the effects of large numbers of compounds in particular tissues/organs and/or cells.
  • the present invention offers the further advantage that any high- throughput and/or microarray system may only require the generation of a single tissue.
  • the methods described herein may find particular application in the study of disease and in particular in testing agents thought to be useful in the treatment of a particular disease and/or condition or in screening and/or identifying agents which are potentially useful as medicaments.
  • the cells for use in the method provided by the fourth aspect of this invention may be derived from a patient known to be suffering from a particular disease. Additionally, or alternatively, the cells may be genetically modified so as to exhibit the symptoms, characteristics or effects of a disease or condition. In this way, once a tissue is established, the tissue may act as a model for a particular disease. Where the resulting tissue is a skin tissue, it may be possible to use cells derived from a patient suffering from a skin disorder and/or to genetically manipulate the cells such that they exhibit the symptoms or effects of a skin disorder.
  • tissue which exhibits the characteristics, symptoms or effects of a particular disease and which tissue further comprises one or more colonies of cells which have been modified so as to include an exogenous component (i.e. a test agent), it may be possible to determine whether and to what extent, a test agent modulates the symptoms, effects and/or characteristics of a particular disease.
  • an exogenous component i.e. a test agent
  • test agents by immobilising as an array one or more test agents at different concentrations, amounts or levels, it may be possible to determine a concentration, level or amount of a particular compound most suited to the treatment of a particular disease.
  • tissue representation of a tissue from which the cells are derived
  • an exogenous component such as a moiety capable of modulating the expression, function and/or activity of a particular gene.
  • the tissue may be generated so as to comprise colonies of cells modified to include an exogenous component capable of modulating the expression, function and/or activity of one or more genes thought to be involved in the pathology of a disease.
  • the exogenous component may comprise one or more genes, or fragments thereof which are capable of being expressed when introduced into a cell (such as those described above). In this way, using only a single assay, it may be possible to investigate, screen and/or analyse the role of the gene or genes in the pathology of certain diseases.
  • diseases and/or conditions characterised by abnormal or aberrant cellular proliferation and/or differentiation may particularly benefit.
  • Other diseases and/or conditions encompassed by the present invention may include those involving signal transduction pathways, homeostasis, differentiation, cell (especially stem cell) development, cell metabolism, aging and/or wound repair.
  • an array of immobilised exogenous components may be used, each comprising an element capable of modulating the expression of one or more genes - in this way it will be possible to generate a tissue comprising a plurality of discrete colonies, the cells of which have been modified (by the inclusion of the exogenous component) such that the expression of one or more gene or genes in each cell of every colony have or has been modulated.
  • the gene or genes to be modulated may be involved in the pathology of the disease to be studied. For example, if a particular type of cancer is to be studied, it may be desirable to modulate the expression of a gene or genes such that the cultured cells exhibit some degree of hyperproliferation.
  • test compound(s) for example an agent or agents thought to be useful in the treatment of a particular disease
  • one or more test compound(s) could be added to the tissue, either directly to a particular colony of transfected cells, or to the entire tissue.
  • Figure 1 Shows immobilised cell-transfecting agents spotted as a 4 x 4 array.
  • Figure 2 A: Viral particles are blotted onto a coated carrier (membrane). After attachment of the particles (2 hrs), keratinocytes are seeded onto the membrane. The cells attach to the carrier and cells transfect themselves. The keratinocytes are cultured until a fully differentiated epidermis has formed. During this differentiation process, the transfected cells will migrate upwards and sidewards and form a colony of cells in which the gene of interest is down- or up regulated.
  • B Viral particles are blotted onto a tissue organ (e.g. dermis, collagen, biological carrier). The same procedure is followed as described in figure IA:
  • Figure 3 Application of 0.5 ul (arrow) onto the filter inserts (A). After incubation time of 30 minutes, the small volume is evaporated and the particles are attached to the coating RetroNectin (B).
  • Figure 4 Keratinocyte mono-layer grown on filter inserts that have been coated and spotted with viral particles (A). Shown are the pores of the filter insert 0.4 ⁇ m (arrows) (B). Magnification 2Ox.
  • FIG. 5 Membranes were coated and blotted with different viral particles containing GFP, Ds-RED or ⁇ -galactosidase (Black arrow). After seeding the cells onto the particles and culturing of the cells till they were fully differentiated, we analysed the expression of GFP- Ds-RED and B-gal in the epidermis. The transfected cells could be seen throughout the membrane in small colonies and in the same order as we have blotted the viral particles ( Figure 5B).
  • Figure 6 Macroscopic overview of the spotted virus particles expressing GFP after two week culture. Two half spots of keratinocytes that are transfected with PGK are visualized (A). One clear spot of keratinocytes that are transfected with CMV virus particles (B). Magnification 4Ox.
  • Figure 7 Cross section of the skin cultures after two weeks of culture. Haematoxylin staining shows the presence of 4 viable cell layers and a stratum corneum (A). Macroscopic illustration (B) and a cross section of a spot expressing B-gal (C). Magnification 4Ox.
  • Figure 8 (A) A 24 wells plate containing 24 membrane inserts (diameter 0.38). (B) Sown is a 12 wells membrane insert that has been coated and onto which 5 micro spots were applied. After application the membrane is left for 2 hrs at room temperature prior seeding the cells of interest.
  • Figure 9 Shown are haematoxylin-eosin stained cross sections of cultured epidermal skin models onto membranes of 0.38 cm (A) and 1.1 cm (B) in diameter. In both membranes the skin cells have been cultured for 14 days at the air-liquid interface.
  • FIG. 10 Cross sections of skin models generated with healthy keratinocytes (A) or a keratinocyte cell line (B).
  • A healthy keratinocytes
  • B stratum granulosum
  • SC stratum comeum
  • Figure 10 Cross sections of skin models generated with healthy keratinocytes (A) or a keratinocyte cell line (B).
  • the cells were seeded onto coated and blotted viral particles expression Ds-Red.
  • the cells were cultured for 14 days at the air liquid interfase. Thereafter the cultures were embedded in Tissue Tek and cross sections were directly analysed with a fluorescence Microscope.
  • the dashed boxes show the colony forming cells expressing Ds-Red. In the near vicinity untransfected cells are observed.
  • FIG. 11 Cross sections of skin models generated with healthy keratinocytes (A) or a keratinocyte cell line (B). In both experiments the cells were seeded onto coated and blotted viral particles expression Ds-Red (white). The cells were cultured for 14 days at the air liquid interface. Thereafter the cultures were embedded in Tissue Tek and cross sections were stained with DAPI (4',6-diamidino-2-phenylindole) to visualize nuclei of the cells (grey) using a fluorescence microscope. The dashed boxes show the colony forming cells expressing Ds-Red. In the near vicinity untransfected cells are observed. Similar observations were made with a keratinocyte cell (B). Materials & Methods lentiviral constructs
  • Lentiviruses used were a gift from Prof. Dr. R. Hoeben LUMC.
  • the viruses tested in this study are (1) LV CMV GFP, ( 987,5 iu/ul); (2) LV PGK GFP, (970 iu/ul); (3) LV PGK dsRed,( 905 iu/ul); (4) LV CMV EFIa LacZ, 3.9xl0E7 HeLa-iu/ml. All viruses were diluted in culture medium, except (4) which was used directly from the stock solution.
  • Viruses 1-3 were diluted at concentrations 500iu / 0,5ul and 50iu / 0,5ul [1:10 dilution] and virus number 3 was diluted at concentration 20.000iu / 0,5ul; 500iu / 0,5ul [1:40 dilution]; 50iu / 0,5ul [1:400 dilution]. Spotting Of Lentivirusses
  • Filterinserts (polyester 0.4 uM, Costar Coming BV, Schiphol-Rijk, The Netherlands) were coated with RetroNectin® (CH-296; Takara, Japan) at a concentration of 50 ⁇ g/mL in PBS. After 2 hours drying a room temperature viral particles (pfu/mL) were spotted using approx. 0.5 ⁇ l. The particles were spotted in an array of 4 x4 spots (see Figure 1). After 30 minutes of drying at room temperature keratinocytes were seeded on the filters.
  • keratinocytes Cultures of normal human keratinocytes (NHK) were established from human breast skin, as described earlier (Ponec et al., 1997). During the isolation procedure, some melanocytes are cultured with the keratinocytes
  • the keratinocyte medium used for the culture of NHK consisted of three parts Dulbecco's modified Eagle medium and one part Ham's F12 medium supplemented with 5% HyClone calf serum (Greiner, Nurtingen, Germany), 1 ⁇ M hydrocortisone, 1 /*M isoproterenol, 0.1 ⁇ M insulin (Sigma-Aldrich, Zwijndrecht, The Netherlands). For experiments, secondary cultures were used.
  • Reconstructed human epidermis was generated on inert filter substrates containing spotted lentiviruses (See above).
  • secondary keratinocyte cultures 500 ⁇ ul containing 1 *10 6 keratinocytes
  • keratinocytes were incubated overnight in keratinocyte medium.
  • the cultures were fed with the same keratinocyte medium but containing 1% serum, 1 xlO "
  • M L-carnitine 1 >d ⁇ ⁇ M L-serine, 1 ⁇ M DL- «-tocopherol-acetate and a lipid supplement containing 25 ⁇ M palmitic acid, 15 ⁇ M linoleic acid, 7 ⁇ M arachidonic acid and 2.4 *10 "5 M bovine serum albumin and cultured under submerged conditions for an additional 5 d. Thereafter, the cultures were lifted to the air-liquid interface and cultured for an additional 2 wk in the same medium except that serum was omitted, the concentration of linoleic acid was increased to 30 mM and 1 ng epidermal growth factor per ml and 50 mg ascorbic acid per ml were added.
  • the skin organs were stained (25 mg 5- Bromo-4-chloro-3-indolyl ⁇ -D-galactopyranoside (sigma); 50 ul MgCl 2 ; 41 mg K- ferficyanide (sigma); 53 mg K-ferrocyanide), by applying 2 ml onto the surface of skin cultures and 3 ml beneath the skin organs.
  • the skin cultures were incubated overnight at 37 0 C in a C ⁇ 2 -free incubator.
  • the skin organs were then rinsed with 100% Ethanol and washed with 100% methyl salicate for 15 minutes. After the washing, the skin cultures were transferred to xylene and further processed for paraffin embedding.
  • paraffin embedded skin cultures were cut in sections (5 ⁇ m) and used for morphological haematoxylin staining and evaluation using a light microscope.
  • RetroNectin To assure the attachment of the virus particles onto a filter insert (2.4cm ⁇ ), we have coated the filter surface with RetroNectin. After the drying process in the incubator, the viral particles were seeded onto the coated surface and incubated for another 30 minutes. After this incubation period, the applied volume (0.5 ⁇ l) evaporated and the virus particles attached to the coating (see Figure 3A).
  • 3DOMs 3-Dimensional Organ Micro-arrays
  • basic skin biology e.g. signal transduction, homeostasis, differentiation and stem cell development
  • aging e.g., aging, wound repair, pathogenesis of skin diseases and skin cancer.
  • 3DOM technology is equally well applicable to other organs and thus provides a platform for similar functional studies in a broad range of human tissues.
  • 3DOM has several crucial advantages.
  • the arrays are compatible with the automated detection of 'high-content phenotypes', using histology and immunocytochemistry and secondly, the scale of 3D0M offers the possibility to use minimal amounts of primary cells making it possible to use patient derived material.
  • References Ponec M, Weerheim A, Kempenaar J, et al The formation of competent barrier lipids in reconstructed human epidermis requires the presence of vitamin C. / Invest Dermatol 1997;109:348-355).

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Abstract

La présente invention concerne la génération de tissus comprenant une ou plusieurs colonies de cellules modifiées, les tissus générés par ces procédés et leurs utilisations dans des procédés de criblage, de test et/ou d’identification de composés et de leurs effets sur une variété d’aspects de la fonction cellulaire. La présente invention concerne en particulier un procédé de génération d’un tissu qui comprend une ou plusieurs colonies de cellules modifiées, ledit procédé comprenant l’étape consistant à introduire dans une ou plusieurs cellules un composant exogène et à maintenir les cellules dans des conditions adaptées pour promouvoir leur croissance, leur prolifération et/ou leur différenciation, pour former un tissu comprenant des colonies de cellules modifiées pour comprendre le composant exogène.
PCT/IB2009/005778 2008-05-30 2009-05-29 Tissus modifiés WO2009144574A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003041568A2 (fr) * 2001-11-15 2003-05-22 University Of Medicine & Dentistry Of New Jersey Matrice tridimensionnelle pour produire des equivalents de tissu vivant

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003041568A2 (fr) * 2001-11-15 2003-05-22 University Of Medicine & Dentistry Of New Jersey Matrice tridimensionnelle pour produire des equivalents de tissu vivant

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
ANDREADIS STELIOS T: "Gene transfer to epidermal stem cells: implications for tissue engineering.", EXPERT OPINION ON BIOLOGICAL THERAPY JUN 2004, vol. 4, no. 6, June 2004 (2004-06-01), pages 783 - 800, XP009122971, ISSN: 1471-2598 *
ANDREADIS STELIOS T: "Gene-modified tissue-engineered skin: the next generation of skin substitutes.", ADVANCES IN BIOCHEMICAL ENGINEERING/BIOTECHNOLOGY 2007, vol. 103, 2007, pages 241 - 274, XP009122939, ISSN: 0724-6145 *
BAJAJ BHARAT ET AL: "Retroviral gene transfer to human epidermal keratinocytes correlates with integrin expression and is significantly enhanced on fibronectin", HUMAN GENE THERAPY, vol. 13, no. 15, 10 October 2002 (2002-10-10), pages 1821 - 1831, XP002546469, ISSN: 1043-0342 *
BAJAJ ET AL: "Efficient Gene Transfer to Human Epidermal Keratinocytes on Fibronectin: In Vitro Evidence for Transduction of Epidermal Stem Cells", MOLECULAR THERAPY, ACADEMIC PRESS, SAN DIEGO, CA, US, vol. 11, no. 6, 1 June 2005 (2005-06-01), pages 969 - 979, XP005002717, ISSN: 1525-0016 *
EL GHALBZOURI A ET AL: "Leiden reconstructed human epidermal model as a tool for the evaluation of the skin corrosion and irritation potential according to the ECVAM guidelines", TOXICOLOGY IN VITRO, ELSEVIER SCIENCE, GB, vol. 22, no. 5, 1 August 2008 (2008-08-01), pages 1311 - 1320, XP022713776, ISSN: 0887-2333, [retrieved on 20080407] *
LEI PEDRO ET AL: "Efficient retroviral gene transfer to epidermal stem cells.", METHODS IN MOLECULAR BIOLOGY (CLIFTON, N.J.) 2008, vol. 433, 2008, pages 367 - 379, XP009122936, ISSN: 1064-3745 *
LELIEVRE ET AL: "The episkin phototoxicity assay (EPA): Development of an in vitro tiered strategy using 17 reference chemicals to predict phototoxic potency", TOXICOLOGY IN VITRO, ELSEVIER SCIENCE, GB, vol. 21, no. 6, 12 August 2007 (2007-08-12), pages 977 - 995, XP022196577, ISSN: 0887-2333 *

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