WO2008076465A1 - Puce à micro-injecteur - Google Patents

Puce à micro-injecteur Download PDF

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Publication number
WO2008076465A1
WO2008076465A1 PCT/US2007/071827 US2007071827W WO2008076465A1 WO 2008076465 A1 WO2008076465 A1 WO 2008076465A1 US 2007071827 W US2007071827 W US 2007071827W WO 2008076465 A1 WO2008076465 A1 WO 2008076465A1
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WIPO (PCT)
Prior art keywords
cells
chip
microinjector
projections
reprogramming
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PCT/US2007/071827
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English (en)
Inventor
Chauncey Sayre
Original Assignee
Primegen Biotech, Llc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Priority claimed from US11/614,899 external-priority patent/US20070142781A1/en
Application filed by Primegen Biotech, Llc filed Critical Primegen Biotech, Llc
Publication of WO2008076465A1 publication Critical patent/WO2008076465A1/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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • 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
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion

Definitions

  • the present invention provides devices and methods for therapeutically reprogramming cells by delivering reprogramming factors directly into the cells. More specifically the present invention provides a microinjector chip device having projections wherein reprogramming compositions are coated or dried onto the projections and are used to deliver the reprogramming compositions into target cells and wherein the cells become therapeutically reprogrammed into pluripotent cells.
  • Currently available methodology for introducing molecules into cells include injecting materials directly into single cells (microinjection) or groups of cells (biolistic approaches) or making the cells more permeable so as to allow uptake of desired molecules from a surrounding medium (micropricking, transfection, electroporation).
  • micropricking a cell membrane is ruptured with a needle and the surrounding medium, containing the injection material, is allowed to diffuse into the cell through the break in the cell membrane.
  • this procedure requires a high degree of manipulative skill by the operator and is very time consuming.
  • Another methodology for inserting injection materials into cells, most often used for the introduction of nucleic acids, such as gene constructs, is the "biolistics” approach wherein high density metallic particles, usually of tungsten or gold, are coated with the nucleic acids and are propelled by gas release at a target cell culture.
  • This approach does not have the precision of microinjection or micropricking but takes the "shotgun” approach which exposes a large number of cells to the injection material with the expectation that some of the cells will take up the injection material. While this method has the potential to reach large numbers of cells relatively easily, it requires expensive equipment, is not efficient and the force of the gas release may harm the target cells.
  • the injection material is suspended in a liquid, and the amount of injection material provided to each cell is dependent on the amount of material which can be suspended in the liquid and the volume of liquid that can be injected into the cell and, second, only a relatively small number of cells is injected, i.e., a low efficiency.
  • a relatively small number of cells i.e., a low efficiency.
  • Therapeutic reprogramming is the induction of somatic cells to dedifferentiate into pluripotent cells by contacting the cells with reprogramming factors that cause epigenetic changes in the target cell.
  • Reprogramming factors such as, but not limited to, proteins, nucleic acids and cellular extracts, most efficiently induce reprogramming when introduced into the cell cytoplasm however such introduction often dilutes the cytoplasm contents. Therefore there is a need for methods of introducing the reprogramming factors into the target cell without diluting the cytoplasm contents. There is also a need for methods that can simultaneously and efficiently introduce reprogramming factors into a plurality of cells.
  • the present invention provides a microinjector chip device for the rapid injection of a large number of cells with minimal operator involvement and minimal dilution of the target molecule with aqueous solutions.
  • the microinjector chip device has a first surface and a second surface and a plurality of projections extending from the first surface about perpendicular to the surface. Injection materials are coated, or deposited, onto the projections allowing for the substantially liquid-free transfer of the injection materials into the cells.
  • the microinjector chip device pierces the target cells and the injection material coated on the projections are deposited within the cells.
  • the invention provides electrically capacitative microinjector chip devices with a plurality of projections that are coated with electrically conductive non-conductive materials deposited on their lateral surfaces with electrically conductive materials deposited at their apical tips.
  • the latter devices allow an electrical charge to be applied to aid in coating and/or injection of materials into cells. Since biological macromolecules like DNA, RNA and protein carry charge, differences in electrical charge are useful in discriminatively injecting cells.
  • Methods of making the microinjector chip devices, coating them with injection materials and delivering the injection materials to target cells are also provided.
  • a microinjector chip for delivery of reprogramming compositions to a plurality of cells comprising a microinjector chip having a first surface and a second surface; and a plurality of projections protruding from said first surface wherein said reprogramming compositions are coated onto at least a subset of said projections.
  • the plurality of projections are coated with an electroconducting material.
  • a method of therapeutically reprogramming a plurality of cells comprising coating the projections of a microinjector chip with an reprogramming composition; bringing the projections of the microinjector chip in close proximity to the plurality of cells; piercing the plurality of cells with the projections; releasing the reprogramming composition into the plurality of cells wherein the reprogramming composition is substantially free of water at the time of injection; and causing the cells to be therapeutically reprogrammed into pluripotent cells.
  • the reprogramming composition is selected from the group consisting of drugs, proteins, nucleic acids, peptides, polysaccharides, viruses, chromosomes, synthetic particles, spores, plasmids, cell organelles, vesicles, liposomes, micelles, and emulsions.
  • the nucleic acid is DNA or RNA.
  • the reprogramming composition further comprises a dye.
  • the coating step is a method selected from the group consisting of freezing, freeze-drying, electrostatic attraction, direct attachment, and biological attachment.
  • the releasing step is induced by vibrating the projections causing the reprogramming composition to be released into the plurality of cells.
  • the vibrating is induced by an integrated circuit disposed on the microinjector chip.
  • the microinjector chip is manufactured from a biocompatible material selected from the group consisting of metals, polymers, quartz, and silica-based materials.
  • Figure 1 depicts a side view of a microinjector chip according to one embodiment of the present invention.
  • Figure 2 depicts one embodiment of the second surface of a microinjector chip according to the teachings of the present invention.
  • Figure 3 depicts one embodiment of the first surface of a microinjector chip according to the teachings of the present invention.
  • Figure 4 depicts the second surface of the microinjector chip of Figure 3.
  • Figure 5 depicts another embodiment of the first surface of a microinjector chip according to the teachings of the present invention.
  • Figure 6 depicts the second surface of the microinjector chip of Figure 4.
  • Figure 7 depicts one embodiment of microinjector chip projections according to the teachings of the present invention.
  • Figure 8 depicts morphology ( Figure 8A) of, and expression of pluripotent markers (Figure 8B) by, HT19 cells transfected with RNAs encoding for Oct-4, Sox2, c-myc and KLf4 according to the teachings of the present invention.
  • NTC no RNA control
  • m mouse embryonic feeder cells
  • N NCCIT cell line
  • 1-4 HT19 cells transfected with RNA and/or reversine.
  • Figure 9 depicts expression of Oct-4 ( Figure 9A) by, and morphology ( Figure 9B) of, HT19 cells transfected with NCCIT total RNA according to the teachings of the present invention.
  • W no RNA control
  • S Sto/c (a mouse embryonic fibroblast, cell line expressing leukemia inhibitory factor)
  • N NCCIT cells
  • 1-13 HT19 cells transfected with NCCIT RNA or control under different conditions (see Table 1 ).
  • Figure 10 depicts expression of pluripotent markers (Figure 10A) by, and morphology (Figure 10B) of, HT33 cells transfected with NCCIT total RNA according to the teachings of the present invention.
  • W no RNA control
  • S Sto/c
  • N NCCIT cells
  • 1 untransfected HT33 cells
  • 2 HT33 transfected with NCCIT RNA.
  • Figure 11 depicts reverse transcriptase-polymerase chain reaction analysis of adult stem cells (HT33) injected with RNA with the microinjector chip according to the teachings of the present invention.
  • Lane W is a template control containing water only;
  • Lane V is a positive control of embryonic stem cells;
  • Lane 1 contains untreated HT-33 cells;
  • Lane 2 contains HT33 cells injected by a first microinjector chip;
  • Lane 3 contains HT33 cells injected by a second microinjector chip.
  • Figure 12 depicts one embodiment of the manufacturing of microinjector chips according to the present invention.
  • the present invention provides a microinjector chip device for the rapid injection of a large number of cells with a target molecule with minimal operator involvement and minimal dilution of the target molecule with aqueous solutions.
  • the microinjector chip device has a first surface and a second surface and a plurality of projections extending from the first surface about perpendicular to the first surface. Injection materials are coated onto the projections allowing the transfer of the substantially liquid-free injection materials into the cells.
  • the microinjector chip device pierces the target cells and the injection material is deposited within the cells. Methods of making the microinjector chip devices, coating them with injection materials and delivering the injection materials to target cells are also provided.
  • substantially free refers to injection materials having less than 10% w/v aqueous components.
  • All of the methods currently available for microinjection of materials into cells have two common drawbacks. First, the injection material is suspended in a carrier liquid, usually an aqueous liquid and second, the methods are relatively inefficient. The amount of injection material provided to each cell is dependent on the maximum amount of material which can be suspended in the liquid and the maximum volume of liquid that can be introduced into a cell. In order to increase the amount of injection material provided to a cell, it is advantageous to limit the amount of liquid. The efficiency of the injected material in producing a desired effect is dependent on each cell within a plurality of cells receiving an effective amount of material.
  • Effective in this context is used to refer to the percentage of cells within a plurality of cells that express a desired effect.
  • Current methods for treating pluralities of cells commonly result in less than 1 % of the cells receiving an effective amount of material to induce a desired effect and often less than 0.1% of is the cells are effectively treated.
  • Inefficiency in current methods presently requires tedious isolation and selection of the effectively treated cells from within the larger plurality of untreated cells. Such methods are not easily amenable to uses in patient treatments.
  • the microinjector chip device of the present invention allows injection materials to be coated onto the microinjector chip's projections and delivered to cells without the diluting effects of carrier liquids.
  • the injection material can be coated onto the microinjector chip projections by a variety of methods including, but not limited to, freezing, freeze drying, direct attachment, electrostatic attraction, or through the use of biological adhesives or fibronectin.
  • the injection material is coated on both the chip body and the projections. In another embodiment, the injection material is coated on only the projections.
  • the projections of the microinjector chip are magnetized such that injection material-coated magnetic microbeads will attach thereto and, after the microinjector chip projections are brought into contact with and pierce the target cells, the magnetic field is released, the microbeads are released into the cell and the microinjector chip is removed.
  • the microbeads are attracted and attached to the projections via electrostatic attraction or a temperature-associated attraction.
  • an electrostatic charge is applied to the projections of the microinjector chip and the projections are then dipped into a solution of injection material such that molecules with the solution are attracted to and attach to the projections.
  • the microinjector chip projections are then brought into contact with and pierce the target cells, the electrostatic attraction is removed by grounding the chip, the injection material is released into the cell and the microinjector chip is removed.
  • the microinjector chip projections are dipped into a concentrated solution of injection material in a sample plate and the injection material is freeze-dried onto the projections. The microinjector chip projections are then brought into contact with and pierce the target cells and the injection materials become rehydrated and are released into the cell after a period of time.
  • the sample plate is preferably coated with a non-stick substance to prevent adherence of the injection material to the plate.
  • Non-stick substances suitable for use on the sample plate are any biocompatible substance including, but not limited to, Teflon ® and silicon-based substances.
  • the sample plate can be coated with a bioactive material, such as but not limited to antibodies, hormone or ligands.
  • sample plate can be used to hold the target cells during the deposition of the injection material.
  • the injection material can be freeze dried onto the microinjector chip projections through a variety of methods.
  • the injection material is freeze dried onto the microinjector chip projections by dipping the projections into a concentrated solution of injection material in the sample plate, freezing the microinjector chip and sample plate together, removing the sample plate and drying the injection material onto the projections.
  • the projections are coated with fibronectin or a biological adhesive prior to dipping into the concentrated solution of injection material in the sample plate. The injection material is then allowed to adhere to the projections and the injection material injected into the target cells.
  • fibronectin or biological adhesive are used to attach the injection material to the projections, the projections may need to be left in contact with the target cells for a period of time from several seconds to several days for the injection materials to become disassociated from the projections and be released into the cells.
  • the biological adhesive is active at temperatures lower than 37°C and when raised to 37°C, as when the projections enter the target cell, release the injection material into the cells.
  • the projections are supercooled then dipped into a concentrated solution of injection material which then freezes onto the projections.
  • the projections are then warmed slightly and the microinjector chip is brought into contact with and pierces the target cells while warming to 37°C to allow the injection material to be released into the target cells.
  • the projections are manufactured from a piezoelectric material and coated with an injection material by any of the foregoing methods.
  • an electrical field is applied to the microinjector chip causing the projections to change shape or elongate, thereby piercing the cells and depositing the injection material into the target cells.
  • another embodiment provides for manufacturing the projections from a thermally active material that changes shape when heated or cooled. By causing the projections to retract when cooled and lengthen when heated, attached injection materials can be introduced and released into cells.
  • the injection material is any material that it is desired to inject into the cell.
  • the injection material can be a purified material or a mixture of materials.
  • the injection material can include, but are not limited to, drugs, peptides, proteins, nucleic acids, polysaccharides, lipids and analogues and conjugates thereof.
  • Proteins can include recombinant and synthetic polypeptides such as DNA methyl-transferases (e.g., DNMTs), histone acetylases, transcription factors (e.g., Oct-4, Sox-2, Klf-4 and the like), oncogene products (e.g., c-Myc, c-Myb, c-Erb and the like), growth regulatory factors (e.g., leukemia inhibitory factor (LIF), fibroblast growth factors (FGFs), glial derived neurotrophic factor (GDNF), epidermal growth factor (EGF) and the like), chromatin proteins (e.g., histone and non-histone proteins) and other epigenetic regulatory factors (e.g., steroid hormone-receptor complexes and the like).
  • DNA methyl-transferases e.g., DNMTs
  • histone acetylases e.g., transcription factors (e.g., Oct-4, Sox-2, K
  • Nucleic acids can include DNAs, cDNAs, RNAs, small interfering RNAs, plasmids, genes, expressible genetic materials, viral DNA or RNA vectors, chromatin, and chromosomes.
  • Polysaccharides can include growth regulatory mucopolysaccharides, ceramides, glycosphingolipids and sialic acid containing lipids.
  • Lipids can include growth regulatory and biologically active phospholipids, phosphoinositides, sulfolipids and intermediates in prostaglandin and arachidonic acid synthesis.
  • the injection material may comprise particles, for example viruses, chromosomes, nucleoli, mitochondria, chloroplasts, Golgi apparatus, endoplasmic reticulum, lysosomes, peroxisomes, centrioles, vacuoles, lipid bilayers, ribosomes, cell organelles, synthetic particles optionally containing or coated with a macromolecule of interest, including, without limitation, timed-release alginates and polysaccharides, spores, plasmids, cell organelles, vesicles, liposomes, micelles and emulsions.
  • the injection material can be a mixture of materials such as a cell extract, a karyoplast extract, a nuclear extract or a cytoplasm extract.
  • the subject extract may be prepared a cell type including, but not limited to, an oocyte, a stem cell (e.g. an embryonic stem cell, an adult stem cell, a fetal stem cell and the like), a cell producing a stem cell growth regulatory factor (e.g., a fibroblast producing LIF and the like).
  • a label for example a dye, such as a fluorescent label, may be added to the injection material to act as a marker to indicate that the injection is successful.
  • the injection material is a pharmaceutically active compound, i.e., a drug.
  • the injection material is a reprogramming composition.
  • Reprogramming compositions are defined as stimulatory factors, including without limitation, chemicals, biochemicals and cellular extracts, either purified components or mixtures.
  • Cellular extracts can be fractionated into nucleic acids, DNA, RNA, lipids or proteins. Additionally, complexes of RNA and protein are also useful for induction of therapeutic reprogramming, e.g. ribonucleoproteins.
  • the microinjector chip projections are coated with an injection material comprising the contents of a first particular cell or cell type and then the injection material is introduced into a second cell or cell type.
  • the injection material is from an embryonic stem cell and the second cell is a quiescent cell from a spermatogonial stem cell population or a somatic cell such as a fibroblast, an adipose cell, a lung parenchymal cell, a gastrointestinal epithelial cell, a connective tissue mesenchymal cell, a neural cell, a cardiac cell, a muscle cell, a kidney cell, a liver cell, a bladder cell, an ovarian cell, a vascular smooth muscle cell and a vascular endothelial cell.
  • FIG. 1 depicts one embodiment of the microinjector chip of the present invention.
  • the microinjector chip 10 comprises a chip body 12 with a flat surface having a first surface 16 and a second surface 18, and the first surface 16 has a plurality of projections 14 suitable for coating with molecules to be injected into target cells.
  • the projections are solid or hollow substantially rigid structures protruding in roughly one direction from the surface of the chip and do not move significantly with respect to the rest of the chip. However, depending on the manufacturing method and the material from which the chip and projections are fabricated, some movement may occur.
  • the chip may be fabricated in any shape suitable for injecting cells including, but not limited to, round, square and rectangular.
  • FIG. 2 depicts one embodiment of microinjector chip 10 having a hollow tube 20 protruding at an angle 22 from the second surface 18.
  • Hollow tube 20 is an optional feature of microinjector chip 10.
  • Hollow tube 20 is a coupling facilitator which allows attachment of the microinjector chip 10 to a micromanipulator or microinjection apparatus.
  • Exemplary, non-limiting, micromanipulator and microinjection apparatuses include those manufactured by Eppendorf (Hamburg, Germany) and Narashige (East Meadow, NY).
  • Hollow tube 20 has a diameter of about 50,000 nm to about 100,000 nm, a length of about 100,000 nm to about 200,000 nm and a wall thickness of about 2,500 nm to about 10,000 nm.
  • angle 22 is between about 45° and about 85°. In one embodiment, angle 22 is between about 55° and about 65°. In another embodiment, angle 22 is about 65°. Furthermore, hollow tube 20 can optionally be present on microinjector chip 15.
  • the projections are between about 5 nm and about 5 ⁇ m in width at their base and between about 10 nm and about 10,000 ⁇ m in length.
  • the diameter of the projections at the point furthest from the surface (the tips) is between about 1 nm to about 5 ⁇ m.
  • the size and length of the projections are based on the type and size of the target cell and on the type of injection material used. Therefore it is within the scope of the present invention to provide microinjector chips with projections of a variety of sizes to accommodate a variety cell types and injection materials.
  • the projections can be spaced on the surface of the chip in any configuration suitable for the particular target cell.
  • the projections are spaced about equidistant from each other and preferably not more than one cell diameter apart from each other.
  • the projections are preferably less than about 15 ⁇ m apart.
  • Figure 7 depicts a microinjector chip 10 having projections 14 on the first surface 16 of the microinjector chip wherein the projections 14 are spaced about equidistant from each other.
  • the microinjector chip projections also generally have a width compatible with the dimension of the cells to be injected.
  • the width of the projection is between about 1 % and about 50% of the cell diameter.
  • cell diameters are from about 10 ⁇ m to about 50 ⁇ m, however the diameter will vary according to the cell type.
  • the microinjector chip projections are hollow.
  • the hollow projections define a tube with a first end and a second end wherein the first end is non-releasably attached to the first surface of the microinjector chip and the second end extends from said first surface substantially perpendicular to the surface.
  • the microinjector chip and projections can be manufactured from a variety of biocompatible metals, polymers or silica-based materials.
  • the chip is fabricated from a heat-conducting material.
  • the chip is fabricated from an electricity-conducting material.
  • the projections are manufactured from the same material as the body of the chip. In yet another embodiment the body of the chip and the projections are manufactured from different materials.
  • Suitable techniques for manufacturing the microinjector chips of the present invention include, but are not limited to, lithography, stamping, LIGA (involving lithography, electroplating and molding), thermoplastic micropattern transfer, resin-based microcasting, micromolding in capillaries (MIMIC), wet isotropic and anisotropic etching, laser assisted chemical etching (LACE), vapor deposition, reactive ion etching (RIE), electron etching and other techniques known within the art of chip fabrication.
  • the microinjector chip is manufactured from quartz and has an integrated circuit placed on the back that aligns with every projection and is used to electronically stimulate the projections to vibrate the injection material off the projections into the cells.
  • Figures 3-6 depict microinjector chips 15 having circuits on the second surface 17 (second surfaces depicted on Figures 4 and 6) such that projections 13 on the first surface 19 are aligned with the circuits and conduct electrical signals to the projections (first surfaces depicted on Figures 3 and 5).
  • Figure 3 depicts a microinjector chip 15 having projections 13 aligned with a circuit 30 comprising electroconducting material disposed in a bent or branched linear pattern on the second surface 17.
  • Figure 4 depicts the second surface 17 of the same microinjector chip as Figure 3 depicting the electroconducting circuit 30 disposed on the second surface 17.
  • Figure 5 depicts an alternative embodiment of microinjector chip 15 wherein projections 13 are aligned with a circuit 50 comprising electroconducting material disposed in a straight linear pattern on second surface 17 and projections 13 are aligned with and extend perpendicular from circuit 50.
  • Figure 6 depicts the second surface 17 of the same microinjector chip as Figure 5 depicting the electroconducting circuit 50 disposed on the second surface 17.
  • an integrated circuit is present at the base of each projection on the same side of the chip as the projections.
  • This style of microinjector can produce a piezoelectric effect to vibrate the injection material off the projection.
  • a thin film deposition of an electroconducting material can be placed on the projection side of the chip and/or on the projections, which can then be electrified to create a piezoelectric effect.
  • circuits may be placed on any material that has a piezoelectric effect, i.e. the chips can be made out of any suitable material to achieve similar results to a quartz chip (i.e. lithium niobate, zinc oxide, silicon oxide, etc.).
  • the microinjector chip of the present invention is particularly suited to the delivery of molecules to cells for therapeutic reprogramming of somatic cells to a pluripotent stem cell-like phenotype.
  • therapeutic reprogramming refers to the process of maturation wherein a non-stem cell such as a somatic cell is exposed to inducing factors according to the teachings of the present invention to commit cells to become either pluripotent or multipotent stem cell-like cells or, alternatively, tissue-specific committed cells of a type different from the tissue of origin of the cells.
  • the reprogramming composition contacts the target cellular machinery directly and effects epigenetic reprogramming.
  • ⁇ pigenetic reprogramming refers to chromosomal changes induced in the target cell that result in silencing of certain genes and activation of other genes by mechanisms that do not involve changes in the DNA nucleotide sequence, as defined further below.
  • the process of therapeutic reprogramming can be performed with a variety of stem cells including, but not limited to, therapeutically cloned cells, hybrid stem cells, embryonic stem cells, fetal stem cells, multipotent post-natal stem cells (adult progenitor cells), adipose-derived stem cells (ADSC) and primordial sex cells.
  • stem cells including, but not limited to, therapeutically cloned cells, hybrid stem cells, embryonic stem cells, fetal stem cells, multipotent post-natal stem cells (adult progenitor cells), adipose-derived stem cells (ADSC) and primordial sex cells.
  • Therapeutic reprogramming takes advantage of the fact that certain stem cells are relatively easily to obtain, such as spermatogonial stem cells, fibroblasts and adipose- derived stem cells, and that reprogramming of these cells can be achieved intrinsically or extrinsically (supra).
  • Therapeutically reprogrammed cells have changed their maturation state to either a more committed differentiated cell lineage or a less committed stem cell-like lineage. Therapeutically reprogrammed cells are therefore capable of repairing or regenerating disease, damaged, defective or genetically impaired tissues.
  • Epigenetics refers to the interactions of genes with their environment which results in a phenotype.
  • Epigenetic modification of DNA includes, effects on chromatin structure, DNA methylation and covalent modifications of histone tails including, but not limited to, acetylation, methylation, phosphorylation, and ubiquitination. Epigenetic modifications can be inherited mitotically and transgenerationally.
  • Intrinsic therapeutic reprogramming uses growth regulatory factors, including without limitation, chemicals, biochemicals and cellular extracts to change the epigenetic programming of cells. These regulatory factors induce, among other results, genomic methylation and/or acetylation changes in the donor DNA and chromatin.
  • Therapeutically reprogrammed cells can be further matured or differentiated into more committed differentiated cell lineages for use in cellular regenerative/reparative therapy.
  • the reprogrammed cells can be subject to maturation and differentiation processes to provide therapeutic cells for uses in treating or replacing damaged cells in pre- and postnatal organs resulting from disease, damage, defect or genetic impairment.
  • HT19 or HT33 Adult human male derived gonadal stem cells (HT19 or HT33) were used as the target cells and transfected with RNA from several sources. Expression of pluripotent markers were then determined in the transfected cells.
  • Group 1 HT19 cells transfected with RNAs encoding for human Oct-4, Sox2, c-myc and KLf4.
  • RNAs encoding for human Oct-4, Sox2, c-Myc, and KLf4 were used as the template for RNA transcription and were obtained from Open Biosystems (Huntsville, AL).
  • Approximately 65 ⁇ g of RNA for each transcribed sequence was pooled together to yield a total amount of RNA of approximately 260 ⁇ g in a total volume of 100 ⁇ l; a concentration of RNA at 2.6 ⁇ g/ ⁇ l.
  • Group 2 HT19 cells transfected with total RNA isolated from a human teratocarcinoma cell line (NCCIT).
  • Group 3 HT33 cells transfected with total RNA isolated from the NCCIT cell line.
  • HT19 or HT33 cells were plated and allowed to attach overnight in FBS- supplemented D-MEM medium.
  • the cells were transfected with the RNAs listed above in an optimized RNA transfection reagent for 2-48 hours and the medium was then replaced with normal complete medium (PM-10TM; as disclosed in co-pending United States Patent Application No. 1 1/488,362 which is incorporated by reference herein for all it contains regarding therapeutic reprogramming and culture media). Images of the cells and samples for RNA analysis were collected at various time points.
  • Figure 8A depicts the morphology of RNA-treated and untreated HT19 cells.
  • Samples 2 and 4 were transfected with Oct-4, Sox2, c-myc, and KLf4 RNA with (sample 4) and without (sample 2) reversine.
  • the transfected cells In contrast to control un-transfected cells which grow in a relatively dispersed non-aggregated manner on the feeder cell substrate, the transfected cells rapidly formed into stable multi-cell aggregates and clumps of cells reminiscent of stem cell cultures.
  • the samples were evaluated for expression of genes associated with pluripotency including Oct-4, Nanog, Sox2, Rex1 , Dppa ⁇ , eRas, and Cripto; the germ-line associated gene Stella; embryonic-associated genes TERT FoxD3, UTF1 , and GDF3; the hematopoietic gene c-kit and a control gene (GAPDH) ( Figure 8B). Only the samples transfected with RNA expressed the pluripotent markers Oct-4 and Sox2. In the RNA- transfected cells TERT and GDF3 were up-regulated.
  • hESC medium D-MEM supplemented with FGF, LIF, non-essential amino acids, glutamax, penicillin, streptomycin, 10% fetal bovine serum, 5 % human plasma A and 5% serum replacement, i.e., with growth additives at the levels disclosed in co-pending United States Patent Application No. 1 1/488,362 which is incorporated by reference herein for all it contains regarding therapeutic reprogramming and culture media.
  • HT33 cells adult human male derived gonadal stem cells
  • Arrays of microinjectors in almost any configuration can be precisely fabricated by micro or nanofabrication processes. These processes can be done on a variety of materials, including silicon, titanium, glass, and polymers. Other materials (such as metals and insulators) can also be integrated onto or within the microchip injector through additional processing steps.
  • One process for fabricating a microinjector chip having an array of needle-like projections in silicon, is described below and in Figure 12.
  • a window area is fabricated that will serve a border to protect the needles (projections) from damage if the array is set down on a surface that could damage the needles.
  • Silicon dioxide or another hard, and etch resistant material
  • PECVD plasma enhanced chemical vapor deposition
  • the thickness of this layer is determined by depth of etching that will be done to form the needle array, so that some of the material will remain after etching is done to form the base of the array.
  • a window area that will enclose the needle array and provide a boundary is now formed in the silicon dioxide.
  • the wafer is first coated with photoresist, a square shaped area is exposed and developed away and then etched by hydrofluoric acid etching. Once etched, the remaining photoresist is dissolved away using organic solvents. The needle array is then formed within the larger window.
  • the array of dots that will serve as a mask for etching the needles is formed on the substrate by photolithographic processing. For several micron wide projections, this can be done using standard contact lithography. For micron and sub-micron sized projections, this can be done using projection lithography systems. For nano-scale sized devices, this can be done by electron beam or interference lithography. In one example, projection lithography is used to form arrays of one to two micron sized dots on a fifteen micron pitch, either in a square or triangular lattice with a negative resist. For silicon fabrication, photoresist is an suitable masking material for dry etching, offering extremely high selectivity greater than 50:1. For etching microprojections in other materials, an intermediate layer may be needed to get desired selectivity. As a non-limiting example, for etching projections in titanium, titanium dioxide or silicon dioxide can be used.
  • the silicon wafer is placed in a plasma-reactive ion etch system to etch the silicon selectively and anisotropically. Based on the parameters used in the etching process, a vertical, or positive or negative sidewall taper can be produced.
  • a vertical, or positive or negative sidewall taper can be produced.
  • the silicon etching in this work used SF 6 , Ar, and C 4 F 8 gases in an inductively coupled plasma to produce the projections.
  • titanium etching CI 2 , Ar, O 2 would be used to produce similar structures.
  • the remaining photoresist mask is removed using solvents.
  • Additional processing steps are incorporated to provide selective area electrical contacts onto the projection tips, with external connection to the outer area of the wafer.
  • the rest of the projections are electrically isolated with an insulating layer.
  • the process described is a self-aligned process that will work on any array of high aspect ratio projections.
  • the process on silicon is described, but can be applied to other materials with slight modification.
  • Silicon is a semiconductor with electrical conductivity much lower than metals. So, first a metal is deposited over the entire silicon microinjector chip using evaporation with fixturing to provide deposition on all sides on the projections (i.e. angle and rotation of the sample with respect to the evaporation source). This could also be done with sputtering or other conformal coating techniques.
  • the metal layers used in this work are titanium/gold/titanium. The first titanium layer is used for adhesion to the silicon. The gold is used to provide superior electrical conductivity. The third titanium layer is used to facilitate the insulator deposition on top of the metal stack as insulators do not adhere well to gold. After metal deposition, silicon dioxide is deposited conformally over the whole sample (except for an area at the edge of the wafer that will be used for outside electrical contact) using PECVD. Once this is done, the self-aligned process is performed.
  • a polymer is spin coated onto the wafer. There will be only very little polymer on the top of the high aspect ratio projections due to the flow of the polymer. But, there will more polymer in between the projections and on large flat areas (such as the border area around the array of projections). Oxygen plasma (or a very short exposure and development) is then applied to the wafer to etch away the polymer uniformly. Since the polymer is thin on the projection tips, this area will be removed for a long time before it is removed from areas in between the projections, thus exposing the top of the pillars in a self-aligned fashion.
  • the silicon dioxide that is covering the metal on the needle tip is selectively removed with a fluorine-based plasma process.
  • the dielectric coating is only removed from the projection tip.
  • the polymer is stripped off the sample using chemical solvents.
  • This fabrication procedure results in an array of micro or nano-sized projections, with electrically conductive tips and electrically isolated sides and areas in between.
  • the electrostatic potential at the tip is applied through the outside connection applied to the metal layer that was deposited on the entire wafer surface.
  • a ground electrode can be deposited on the wafer back-side if desired, by any deposition technique. As noted earlier, if the wafer itself is metallic, such as titanium, then the thin-metal deposition step can be eliminated and the wafer back-side can be electrically isolated through the addition of an insulating layer on the wafer back-side.

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Abstract

L'invention concerne une puce à micro-injecteur et des procédés associés, pour micro-injecter dans une pluralité de cellules, des matériaux d'injection, la puce à micro-injecteur comprenant une pluralité de projections dépassant en parallèle d'une surface supérieure de la puce à micro-injecteur et étant perpendiculaires à celle-ci.
PCT/US2007/071827 2006-12-21 2007-06-21 Puce à micro-injecteur WO2008076465A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US11/614,899 2006-12-21
US11/614,899 US20070142781A1 (en) 2005-12-21 2006-12-21 Microinjector chip
USPCT/US2006/062522 2006-12-21
PCT/US2006/062522 WO2007076458A1 (fr) 2005-12-21 2006-12-21 Puce a micro-injecteur

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WO2008076465A1 true WO2008076465A1 (fr) 2008-06-26

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5457041A (en) * 1994-03-25 1995-10-10 Science Applications International Corporation Needle array and method of introducing biological substances into living cells using the needle array
WO1996010630A1 (fr) * 1994-09-30 1996-04-11 Rutgers, The State University Introduction directe de substances etrangeres dans des cellules
WO2000005339A1 (fr) * 1998-07-22 2000-02-03 The Secretary Of State For Defence Transfert de matieres dans des cellules utilisant du silicium poreux
US20040063100A1 (en) * 2002-09-30 2004-04-01 Wang Chung Lin Nanoneedle chips and the production thereof
WO2007003398A2 (fr) * 2005-07-01 2007-01-11 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Systeme d'electrodes, son utilisation et procede pour le produire
US20070142781A1 (en) * 2005-12-21 2007-06-21 Sayre Chauncey B Microinjector chip

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5457041A (en) * 1994-03-25 1995-10-10 Science Applications International Corporation Needle array and method of introducing biological substances into living cells using the needle array
WO1996010630A1 (fr) * 1994-09-30 1996-04-11 Rutgers, The State University Introduction directe de substances etrangeres dans des cellules
WO2000005339A1 (fr) * 1998-07-22 2000-02-03 The Secretary Of State For Defence Transfert de matieres dans des cellules utilisant du silicium poreux
US20040063100A1 (en) * 2002-09-30 2004-04-01 Wang Chung Lin Nanoneedle chips and the production thereof
WO2007003398A2 (fr) * 2005-07-01 2007-01-11 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Systeme d'electrodes, son utilisation et procede pour le produire
US20070142781A1 (en) * 2005-12-21 2007-06-21 Sayre Chauncey B Microinjector chip
WO2007076458A1 (fr) * 2005-12-21 2007-07-05 Primegen Biotech Llc Puce a micro-injecteur

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