EP1836293A2 - Surface solide comprenant un polymere cationique degradable immobilise afin de transfecter des cellules eucaryotes - Google Patents

Surface solide comprenant un polymere cationique degradable immobilise afin de transfecter des cellules eucaryotes

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Publication number
EP1836293A2
EP1836293A2 EP05854197A EP05854197A EP1836293A2 EP 1836293 A2 EP1836293 A2 EP 1836293A2 EP 05854197 A EP05854197 A EP 05854197A EP 05854197 A EP05854197 A EP 05854197A EP 1836293 A2 EP1836293 A2 EP 1836293A2
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European Patent Office
Prior art keywords
transfection
cell
formula
degradable
cells
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EP05854197A
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German (de)
English (en)
Inventor
Yasunobu Tanaka
Chris P. Castello
Lei Yu
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Nitto Denko Corp
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Nitto Denko Corp
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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
    • C12N5/0068—General culture methods using substrates
    • 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
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/30—Synthetic polymers

Definitions

  • Embodiments of the invention relate to devices and methods for cell transfection.
  • embodiments of the invention are directed to a cell transfection formula and to a cell culture device that has been treated with the transfection formula.
  • the treated cell culture device can be stored at room temperature and provides a transfection method that is simple and quick.
  • Gene transfection methods can be used to introduce nucleic acids into cells and are useful in studying gene regulation and function. High throughput assays that can be used to screen large sets of DNAs to identify those encoding products with properties of interest which are particularly useful. Gene transfection is the delivery and introduction of biologically functional nucleic acids into a cell, particularly a eukaryotic cell, in such a way that the nucleic acid retains its function within the cell. Gene transfection is widely applied in studies related to gene regulation, gene function, molecular therapy, signal transduction, drug screening, and gene therapy studies. As the cloning and cataloging of genes from higher organisms continues, researchers seek to discover the function of the genes and to identify gene products with desired properties. This growing collection of gene sequences requires the development of systematic and high-throughput approaches to characterizing gene products and analyzing gene function, as well as other areas of research in cell and molecular biology.
  • Non-viral carriers are simpler and more cost effective in comparison to preparation of viral carriers, making synthetic gene carriers desirable as transfection reagents, particularly for in vitro studies.
  • Most non-viral vectors mimic important features of viral cell entry in order to overcome cellular barriers, which are meant to prevent infiltration by foreign genetic material.
  • Non-viral gene vectors based on a gene carrier backbone, can be classified as a) lipoplexes, b) polyplexes, and c) lipopolyplexes.
  • Lipoplexes are assemblies of nucleic acids with a lipidic component, which is usually cationic. Gene transfer by lipoplexes is called lipofection.
  • Polyplexes are complexes of nucleic acids with cationic polymer.
  • non-viral transfection reagents are synthetic cationic molecules and have been reported to "coat" the nucleic acid by interaction of the cationic sites on the cation and anionic sites on the nucleic acid.
  • the positively-charged DNA-cationic molecule complex interacts with the negatively charged cell membrane to facilitate the passage of the DNA through the cell membrane by non-specific endocytosis. (Schofield, Brit. Microencapsulated. Bull, 51(1):56-71 (1995)).
  • the cells are seeded on cell culture devices 16 to 24 hours before transfection.
  • the transfection reagent (such as a cationic polymer carrier) and DNA are usually prepared in separate tubes, and each respective solution is diluted in medium (containing no fetal bovine serum or antibiotics). The solutions are then mixed by carefully and slowing adding one solution to the other while continuously vortexing the mixture. The mixture is incubated at room temperature for 15-45 minutes to allow complex formation between the transfection reagent and the DNA and to remove residues of serum and antibiotics. Prior to transfection, the cell culture medium is removed and the cells are washed with buffer. The solution containing the DNA-transfection reagent complexes is added to the cells, and the cells are incubated for about 3-4 hours. The medium containing the transfection reagent is then be replaced with fresh medium. The cells are finally analyzed at one or more specific time point(s). This is obviously a time consuming procedure, particularly when the number of samples to be transfected is very large.
  • transfection is a lengthy and technically difficult procedure. Generally, three steps are required: 1) cells are seeded in a cell culture plate or dish and incubated until sufficient confluence is achieved; 2) transfection reagent / nucleic acid complexes are prepared; and 3) nucleic acids of interest are added along with the transfection reagent and further incubation is carried out. Two incubation periods are needed and typically it takes more than two days to complete all the steps. In contrast, embodiments of the present invention provide a simple procedure that involves only a single incubation step. A cell culture device, which has previously been coated with a transfection reagent, allows transfection by adding the nucleic acid of interest and the cell culture in succession.
  • the degradable cationic polymer is made by linking cationic compounds or oligomers with degradable linkers.
  • the transfection agent may comprise both a degradable cationic polymer and a non-degradable cationic polymer.
  • the ratio of the non-degradable cationic polymer to the degradable cationic polymer is 1:0.5 to 1:20 (non-degradable : degradable) by weight.
  • the transfection reagent includes a plurality of cationic molecules and at least one degradable linker molecule connecting said cationic molecules in a branched arrangement, wherein said cationic molecules are selected from : (i) a cationic compound of formula (A) or (B) or a combination thereof:
  • R 1 is a hydrogen atom, an alkyl of 2 to 10 carbon atoms, another Formula A, or
  • R 4 is a hydrogen atom, an alkyl of 2 to 10 carbon atoms, another Formula A, or Formula B;
  • the linker molecule is 1,3-butanediol diacrylate, 1,4- butanediol diacrylate, 1,6-hexanediol diacrylate, 2,4-pentanediol diacrylate, 2-methyl-2,4- pentanediol diacrylate, 2,5-dimethyl-2,5-hexanediol diacrylate, poly(ethylene glycol) diacrylate, trimethylolpropane triacrylate, pentaerythritol tetraacrylate, di(trimethylolpropane) tetraacrylate, dipentaerythritol pentaacrylate, or a polyester with at least three acrylate or acrylamide side groups.
  • the molecular weight of the polymer is from 500 da to
  • the molecular weight of the cationic compound or oligomer is from 50 da to 10,000 da. In preferred embodiments, the molecular weight of the linker molecule is from 100 da to 40,000 da.
  • the solid support is a dish bottom, a multi-well plate, or a continuous surface.
  • the cell is a mammalian cell. hi some preferred embodiments, at least some of the cells undergo cell division, hi some preferred embodiments, the cell is a transformed or primary cell. In some preferred embodiments, the cell is a somatic or stem cell. In some preferred embodiments, the cell is a plant cell. Further aspects, features and advantages of this invention will become apparent from the detailed description of the preferred embodiments which follow.
  • Embodiments of the invention are directed to a transfection device and method which is simple, convenient, and efficient compared to conventional transfection assays.
  • a transfection device is made according to methods described herein by affixing a transfection reagent on the solid surface of a cell culture device. By using this device, researchers need only add a nucleic acid or other biomolecule to be transfected and cells to the surface of the cell culture device. There is no need to pre-mix the DNA or biomolecule with a transfection reagent. This removes a key timing-consuming step, which is required by conventional transfection procedures. Only approximately 40 minutes is required to complete the entire transfection process for 10 samples, compared to 2 to 5 hours or more required by current methods. This is particularly advantageous for high throughput transfection assays, in which hundreds of samples are tested at a time.
  • transfection reagents are simply coated onto the surface of a cell culture device, which can be easily commercialized and mass-produced.
  • An incubation period for a predetermined time allows the biomolecule and the transfection reagent(s) to form a complex for uptake by cells in the next step.
  • Cells are then seeded on the surface of the cell culture device and incubated, without the necessity of changing the medium, and the cells are analyzed. Changing medium during the transfection procedure is unnecessary.
  • the composition containing the transfection agent can be affixed to any suitable surface.
  • the surface can be glass, plastics (such as polytetrafluoroethylene, polyvinylidenedifluoride, polystyrene, polycarbonate, polypropylene), silicon, metal (such as gold), membranes (such as nitrocellulose, methylcellulose, PTFE or cellulose), paper, biomaterials (such as protein, gelatin, agar), tissues (such as skin, endothelial tissue, bone, cartilage), or minerals (such as hydroxylapatite, graphite).
  • the surfaces may be slides (glass or poly-L-lysine coated slides) or wells of a multi-well plate.
  • the nucleic acid/transfection reagent complexes create a medium for use in high throughput microarrays, which can be used to study hundreds to thousands of nucleic acids, or other biomolecules at the same time, hi an alternative embodiment, the transfection reagents can be affixed on the surface of the transfection device in discrete, defined regions to form a microarray of transfection reagents.
  • biomolecules such as nucleic acids, which are to be introduced into cells, are spread on the surface of the transfection device and incubated with the transfection reagent microarray. This method can be used in screening transfection reagents or other delivery reagents from thousands of compounds. The results of such a screening method can be examined through computer analysis.
  • the transfection device is stored in a material suitable for packaging which may be plastic (e.g., cellophane), an elastomeric material, thin metal, Mylar®, or other polyester film material.
  • the storage may be with or without oxygen and/or carbon dioxide absorbers.
  • the transfection plates prepared as described herein may be stored for at least 5 months at room temperature with retention of significant cell- transfecting activity.
  • the transfection reagent is preferably a cationic compound which can introduce biomolecules, such as nucleic acids into cells.
  • Preferred embodiments use cationic oligomers, such as low molecular weight polyethyleneimine (PEI).
  • the transfection agent is a degradable cationic polymer.
  • the transfection agent includes a cell-targeting or an intracellular-targeting moiety and/or a membrane- destabilizing component, as well as delivery enhancers. In general, delivery enhancers fall into two categories. These are viral carrier systems and non- viral carrier systems. As human viruses have evolved ways to overcome the barriers to transport into the nucleus discussed above, viruses or viral components are useful in transport of nucleic acid into cells.
  • the degradable polymers may be conjugated to or associated with a viral or non-viral protein to enhance transfection efficiency.
  • a viral or non-viral protein for example, vesicular stomatitis virus G protein (VSVG) and other peptides or proteins which are known to those of skill in the art may be added to the polymers in order to improve transfection efficiency.
  • VSVG vesicular stomatitis virus G protein
  • other peptides or proteins which are known to those of skill in the art may be added to the polymers in order to improve transfection efficiency.
  • HA-peptide hemagglutinin peptide
  • This viral peptide facilitates transfer of biomolecules into cells by endosome disruption. At the acidic pH of the endosome, this protein causes release of the biomolecule and carrier into the cytosol.
  • Non- viral delivery enhancers may be either polymer-based or lipid-based. They are generally polycations which act to balance the negative charge of the nucleic acid. Polycationic polymers have shown significant promise as non- viral gene delivery enhancers due in part to their ability to condense DNA plasmids of unlimited size and to safety concerns with viral vectors. Examples include peptides with regions rich in basic amino acids such as oligo-lysine, oligo-arginine or a combination thereof and polyethylenimine (PEI). These polycationic polymers facilitate transport by condensation of DNA. Branched chain versions of polycations such as PEI and Starburst dendrimers can mediate both DNA condensation and endosome release (Boussif, et al.
  • PEI polyethylenimine
  • Another means to enhance delivery is to design a ligand on the transfection reagent.
  • the ligand must have a receptor on the cell that has been targeted. Biomolecule delivery into the cell is then initiated by receptor recognition. When the ligand binds to its specific cell receptor, endocytosis is stimulated.
  • Examples of ligands which have been used with various cell types to enhance biomolecule transport are galactose, transferrin, the glycoprotein asialoorosomucoid, adenovirus fiber, malaria circumsporozite protein, epidermal growth factor, human papilloma virus capsid, fibroblast growth factor and folic acid.
  • the bound ligand is internalized through a process termed potocytosis, where the receptor binds the ligand, the surrounding membrane closes off from the cell surface, and the internalized material then passes through the vesicular membrane into the cytoplasm (Gottschalk, et al. (1994) Gene Ther 1:185-191).
  • Various agents have been used for endosome disruption. Besides the HA-protein described above, defective-virus particles have also been used as endosomolytic agents (Gotten, et al. (My 1992) Proc. Natl. Acad. Sci. USA vol. 89: pages 6094-6098).
  • Non- viral agents are either amphiphillic or lipid-based.
  • toxins such as Diptheria toxin and Pseudomonas exotoxin have been utilized as components of chimeric proteins that can be incorporated into a gene/gene carrier complex (Uherek, et al.(1998) J Biol. Chem. vol. 273: 8835-8841). These components promote shuttling of the nucleic acid through the endosomal membrane and back through the endoplasmic reticulum.
  • the linker molecules may contain biologically, physically or chemically cleavable bonds, such as hydrolysable bonds, reducible bonds, a peptide sequence with enzyme specific cleavage sites, pH sensitive, or sonic sensitive bonds.
  • the degradation of these polymers may be achieved by methods including, but not limited to hydrolysis, enzyme digestion, and physical degradation methods, such as optical cleavage (photolysis).
  • One of the advantages of the degradable cationic polymers described herein is that degradation of the polymers is controllable in terms of rate and site of polymer degradation, based on the type and structures of the linkers.
  • the transfection reagent includes a plurality of cationic molecules and at least one degradable linker molecule connecting said cationic molecules in a branched arrangement.
  • Cationic oligomers such as low molecular weight polyethyleneimine (PEI), low molecular weight poly(L-lysine) (PLL), low molecular weight chitosan, and low molecular weight PAMAM dendrimers, can be used to make the polymers described herein. Furthermore, any molecule containing amines with more than three reactive sites can be used.
  • PEI low molecular weight polyethyleneimine
  • PLL low molecular weight poly(L-lysine)
  • PAMAM dendrimers low molecular weight PAMAM dendrimers
  • Cationic oligomers maybe selected from, but are not limited to:
  • Formula B Formula A wherein R 1 is a hydrogen atom, an alkyl of 2 to 10 carbon atoms, another Formula A, or Formula B; R 2 is a straight chain alkylene group of the formula: -(CH 2 ) a - wherein a is an integer number from 2 to 10; R 3 is a straight chain alkylene group of the formula: -(Q,H 2b )- wherein b is an integer number from 2 to 10;
  • R 5 is a hydrogen atom, an alkyl of 2 to 10 carbon atoms, another Formula A, or Formula B;
  • cationic molecules examples include, but are not limited to, the cationic molecules shown in Table 1.
  • Table 1 Structures of cationic compounds and oligomers according to preferred embodiments of the invention
  • Cationic polymers used herein may include primary or secondary amino groups, which can be conjugated with active ligands, such as sugars, peptides, proteins, and other molecules.
  • lactobionic acid is conjugated to the cationic polymers.
  • the galactosyl unit provides a useful targeting molecule towards hepatocyte cells due to the presence of galactose receptors on the surface of the cells.
  • lactose is conjugated to the degradable cationic polymers in order to introduce galactosyl units onto the polymer.
  • Reactive residues of the linker molecules have been illustrated in Table 2, however these examples are not limiting to the scope of the invention.
  • Reactive residues may be selected from, but are not limited to, acryloyl, maleimide, halide, carboxyl acylhalide, isocyanate, isothiocyanate, epoxide, aldehyde, sulfonyl chloride, and N-hydroxysuccinimide ester groups or combinations thereof.
  • Table 2 Structures of biodegradable linker molecules used in preferred embodiments of the invention
  • Acrylate linkers are much cheaper than disulfide-containing linkers, because synthesis of the disulfide-containing linkers is more difficult. Acrylate linkers can be hydro lysable in any water solution. Therefore a polymer containing acrylate linkers can be degraded in various environments as long as it contains water. Thus, polymers containing acrylate linkers have broad applications compared to disulfide-linker-containing polymers, hi addition, the degradation rate of polymers with disulfide-linkers are usually the same, but the degradation rate of polymers synthesized with acrylate linkers can vary depending on the different acrylate linkers used.
  • the transfection agent may comprise both a degradable cationic polymer and a non-degradable cationic polymer.
  • the ratio of the non-degradable cationic polymer to the degradable cationic polymer is preferably from 1:0.5 to 1:20 (non-degradable : degradable) by weight, and more preferably from 1 :2 to 1 : 10 by weight.
  • the concentration of transfection reagent present in the mixture depends on the transfection efficiency and cytotoxicity of the reagent. Typically there is a balance between transfection efficiency and cytotoxicity. At concentrations in which a transfection reagent is most efficient, while keeping cytotoxicity at an acceptable level, the concentration of transfection reagent is at the optimal level.
  • the concentration of transfection reagent will generally be in the range of about 1.0 ⁇ g/ml to about 1000 ⁇ g/ml. In preferred embodiments, the concentration is from about 10 ⁇ g /ml to about 600 ⁇ g/ml.
  • the DNA can also be linear fragment with a promoter sequence (such as CMV promoter) at the 5' end of the cDNA to be expressed and a poly A site at the 3' end.
  • a promoter sequence such as CMV promoter
  • These gene expression elements allow the cDNA of interest to be transiently expressed in mammalian cells.
  • the DNA is a single strand oligodeoxynucleotide (ODN), for example antisense ODN, it can be introduced into cells to regulate target gene expression.
  • ODN oligodeoxynucleotide
  • the nucleic acid may be single stranded (antisense RNA and ribozyme) or double stranded (RNA interference, SiRNA).
  • the RNA is modified in order to increase the stability of RNA and improve its function in down regulation of gene expression.
  • any appropriate cell culture media may be used including, but not limited to, Minimum Essential Eagle, F- 12 Kaighn's Modification medium, or RPMI 1640 medium.
  • the transfection reagent is evenly affixed on the slide, the nucleic acid solution can be spotted onto discrete locations on the slide. Alternatively, transfection reagents may be spotted on discreet locations on the slide, and the nucleic acid solution can simply be added to cover the whole surface of the transfection device. If the transfection reagent is affixed on the bottom of multi-well plates, the nucleic acid solution is simply added into different wells by multi-channel pipette, automated device, or other delivery methods which are well known in the art.
  • the resulting product (a surface bearing biomolecules and plated cells) is maintained under conditions that result in entry of the nucleic acids of interest into the plated cells.
  • the cells are mixed with the biomolecule or nucleic acid.
  • the cell/biomolecule mixture is then added to the transfection device and incubated at room temperature.
  • Suitable cells for use according to the methods described herein include prokaryotes, yeast, or higher eukaryotic cells, including plant and animal cells, especially mammalian cells.
  • eukaryotic cells such as mammalian cells (e.g., human, monkey, canine, feline, bovine, or murine cells), bacterial, insect or plant cells, are plated onto the transfection device, which is coated with transfection reagent and nucleic acids of interest, in sufficient density and under appropriate conditions for introduction/entry of the nucleic acids of interest into the eukaryotic cells and either expression of the DNA or interaction of the biomolecule with cellular components.
  • the cells may be selected from hematopoietic cells, neuronal cells, pancreatic cells, hepatic cells, chondrocytes, osteocytes, or myocytes. The cells can be fully differentiated cells or progenitor/stem cells.
  • PEI-600 in 25 ml of methylene chloride were added by using pipette or syringe. 2.09 g of
  • PDODA was quickly added to the above PEI-600 solution with stirring.
  • the reaction mixture was stirred for 4 hours at room temperature (20° C). Then, the reaction mixture was neutralized by adding 50 ml of 2M HCl. The white precipitate was centrifuged, washed with methylene chloride, and dried at room temperature under reduced pressure.
  • L-PEI Linear polyethyleneimine
  • lipid based polymers were used for transfecting plasmid DNA into mammalian cells in vitro to evaluate the transfection efficiency.
  • L-PEI based polymer jet PEI (Qbiogene) transfection reagent was used for L-PEI based polymer.
  • Lipofectamine2000 (Invitrogen) and N-[l-(2,3-dioleoyloxy)propyl]-N,N,N- trimethylammonium salts (DOTAP; Sigma-Aldrich) were employed as lipid based polymers.
  • Degradable cationic polymer and DOTAP were dissolved in methanol, and jet PEI and Lipofectamine2000 were diluted by deionized water.
  • Flat bottom 96-well cell culture plates (bottom surface: 0.32 cm 2 per each well; BD Biosciences) were treated with these polymer solutions.
  • Transfection with Transfectable Cell Culture Device for 293 Cells 25 or 50 ng of pEGFP-Nl plasmid (purchased from Clontech) in 25 ⁇ l of opti- MEM I (hivitrogen) was added in each well and kept at room temperature for 25 minutes. Then, 5 X 10 4 of 293 cells in 100 ⁇ l of Dulbecco's modified Eagle Medium (DMEM) (Invitrogen) with 10% calf serum ( ⁇ nvitrogen) were added and incubated at 37°C in 7.5% of CO 2 . After 24 to 36 hrs. incubation, transfection efficiency was estimated by observing EGFP fluorescence by using epifluorescent microscope (1X70, Olympus).
  • DMEM Dulbecco's modified Eagle Medium
  • Figure 2 shows percentages of EGFP-positive cells after transfection. High transfection efficiencies were allocated between 2.5 to 5.0 ⁇ g per well (thus, 7.8 to 16 ⁇ g/cm 2 ) of degradable cationic polymer with 0.25 and 0.5 ⁇ g per well (thus, 0.78 to 1.6 ⁇ g/cm 2 ) of plasmid DNA.
  • transfection efficiency with plasmid DNA carrying luciferase gene was tested periodically.
  • the procedure for transfectable cell culture devices was as described in Example 3 except the plasmid DNA was different.
  • Luciferase activity of cells were determined by using a Dynex MLX Microtiter ® plate luminometer and Luciferase Assay System (Promega Corp. Madison, WI USA) to determine transfection efficiency.
  • Figures 4, 5 and 6 show change of transfection efficiencies after storage at 25° C with O 2 and/or CO 2 absorbing materials in Mylar Bags. There was no obvious decrease of transfection efficiency after 5 month storage. Moreover, even when cell culture devices were kept at 25 ° C in Mylar Bags without O 2 and/or CO 2 absorbing materials, transfection efficiency was stable after 5 month and still quite high (Figure 7).
  • the cell culture devices of this invention are quite stable at room temperature. The device can be stored without special storage conditions.
  • Non-degradable polymer was prepared as follows: Approximately 5 g of polyethlenimine (Aldrich, product number: 408727) was dissolved in 50 ml of dichloromethane, then 100 ml of 2.0M hydrogen chloride in diethyl ether (Aldrich, product number: 455180) was added and mixed well to form polymer hydrochloride. Then, the polymer hydrochloride was collected by centrifuge, and rinsed with 150 ml of diethyl ether. This rinse with diethyl ether was carried out twice. The resultant precipitation after the rinse was dried under vacuum condition at room temperature for 3 hours. Then, the dried powder was stored at 4 0 C with desiccant until use.
  • Example 8 Preparation of 96-well Transfectable Cell Culture Device with Degradable Cationic
  • Degradable cationic polymer was prepared as indicated in Example 1.
  • Non- degradable cationic polymer was obtained as described in Example 7. Both polymers were dissolved in methanol and mixed together to make a coating solution. The final concentration of each polymer was: Degradable cationic polymer; 40 ⁇ g/ml, and Non- degradable cationic polymer; 10 ⁇ g/ml. Then, flat bottom 96-well cell culture plates (bottom surface: 0.32 cm 2 per each well; BD Biosciences) were treated with the coating solution. Actually, 25 ⁇ l of the coating solution was dispensed in each well, and dried under vacuum condition to remove methanol.
  • Degradable cationic polymer was prepared as indicated in Example 1.
  • Non- degradable cationic polymer was obtained as described in Example 7. Both polymers were dissolved in methanol and mixed together to make a coating solution. The final concentration of each polymer was: Degradable cationic polymer; 80 ⁇ g/ml, and Non- degradable cationic polymer; 10 ⁇ g/ml. Then, flat bottom 12-well cell culture plates (bottom surface: 3.8 cm 2 per each well; BD Biosciences) were treated with these polymer solutions. 100 ⁇ l of the coating solution was dispensed in each well, and dried under vacuum condition to remove methanol.
  • Example 10 Preparation of 6-well Transfectable Cell Culture Device with Degradable Cationic Polymer and Non-degradable Cationic Polymer
  • Degradable cationic polymer was prepared as indicated in Example 1.
  • Non- degradable cationic polymer was obtained as described in Example 7. Both polymers were dissolved in methanol and mixed together to make a coating solution. The final concentration of each polymer was: Degradable cationic polymer; 80 ⁇ g/ml, and Non- degradable cationic polymer; 10 ⁇ g/ml.
  • flat bottom 6-well cell culture plates bottom surface: 9.6 cm 2 per each well; BD Biosciences
  • Mammalian cells were incubated in 10-cm cell culture dishes, rinsed with phosphate-buffered saline, and treated with trypsin solution. Then, the trypsinized cells were diluted in appropriate cell culture medium with serum to prepare a cell suspension.
  • the cell density used in this example is shown in Table 4.
  • pEGFP-Nl plasmid was diluted in opti-MEM, and the final concentration was adjusted to 10 ⁇ g/ml. Then, 25 ⁇ l of the plasmid solution was added in each well of the 96- well transferable cell culture device prepared as indicated in Example 8, and kept at room temperature for 25 minutes.
  • Table 4 indicates the percentage of the cells with EGFP fluorescence in various mammalian cell lines.
  • the 96-well transfectable cell culture device in this invention transfected various mammalian cell lines with high efficiency.
  • Example 12 Transfection with 12-well Transfectable Cell Culture Devices Prepared with Degradable and Non-degradable Cationic Polymers
  • Mammalian cells were incubated in 10-cm cell culture dishes, rinsed with phosphate-buffered saline, and treated with trypsin solution. Then, the trypsinized cells were diluted in appropriate cell culture medium with serum to prepare cell suspension.
  • the cell density used in this example is shown in Table 4.
  • pEGFP-Nl plasmid was diluted in opti-MEM, and the final concentration was adjusted to 5 ⁇ g/ml. Then, 200 ⁇ l of the plasmid solution was added in each well of the 12- well transfectable cell culture device prepared as indicated in Example 9, and kept at room temperature for 25 minutes.
  • Table 4 indicates the percentage of the cells with EGFP fluorescence in various mammalian cell lines.
  • the 12-well transfectable cell culture device in this invention transfected various mammalian cell lines with high efficiency.
  • Example 13
  • Mammalian cells were incubated in 10-cm cell culture dishes, rinsed with phosphate-buffered saline, and treated with trypsin solution. Then, the trypsinized cells were diluted in appropriate cell culture medium with serum to prepare cell suspension.
  • the cell density used in this example is shown in Table 4.
  • pEGFP-Nl plasmid was diluted in opti-MEM, and the final concentration was adjusted to 5 ⁇ g/ml. Then, 400 ⁇ l of the plasmid solution was added in each well of the 6- well transfectable cell culture device prepared as indicated in Example 10, and kept at room temperature for 25 minutes. Then, 2 ml of the cell suspension was added in the well, and incubated at 37°C in 7.5% of CO 2 . After 36 to 48-hour incubation, transfection efficiency was estimated by observing EGFP fluorescence by using epifluorescent microscope (1X70, Olympus).
  • Table 4 indicates the percentage of the cells with EGFP fluorescence in various mammalian cell lines.
  • the 6-well transfectable cell culture device in this invention transfected various mammalian cell lines with high efficiency.
  • HeLa 70 80 70 1.0-2.0x10 s 1.0x10 s 0.5x10 s
  • CV- 1 20-30 30 20-30 1.0x10 s 1.5x10 5 1.5x10 s

Abstract

L'invention concerne un dispositif de transfection/culture cellulaire qui comprend un support solide recouvert d'un cation polymère dégradable en tant que réactif de transfection. Ce dispositif de transfection/culture cellulaire est stocké de manière pratique à température ambiante jusqu'à son utilisation. Pour effectuer facilement la transfection cellulaire, l'acide nucléique d'intérêt et les cellules à transfecter sont ajoutés au dispositif de transfection/culture. La transfection cellulaire est achevée en moins d'une heure au moyen du dispositif de transfection/culture selon l'invention.
EP05854197A 2004-12-17 2005-12-14 Surface solide comprenant un polymere cationique degradable immobilise afin de transfecter des cellules eucaryotes Withdrawn EP1836293A2 (fr)

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KR20130010036A (ko) 2013-01-24
AU2005316501A1 (en) 2006-06-22
AU2005316501B2 (en) 2010-10-21
KR20070101859A (ko) 2007-10-17
WO2006066001A2 (fr) 2006-06-22
JP4739352B2 (ja) 2011-08-03
CN101124316A (zh) 2008-02-13
CA2590820A1 (fr) 2006-06-22
KR101263173B1 (ko) 2013-05-13
JP2008523810A (ja) 2008-07-10

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