초록 ▼

We disclose methods of sorting or separating mixtures of living cells (e.g., eukaryotic, prokaryotic, mammalian, pathogenic, bacterial, viral, etc.). We perform our methods by activating cell-selective photophoric labels, which photosensitize and chemically reduce a photosensitive metal compound to

We disclose methods of sorting or separating mixtures of living cells (e.g., eukaryotic, prokaryotic, mammalian, pathogenic, bacterial, viral, etc.). We perform our methods by activating cell-selective photophoric labels, which photosensitize and chemically reduce a photosensitive metal compound to form metal grains, particles or crystals. The metal adheres to the cells and forms the basis for sorting or separating different cell types. Photophoric labels may include chemiluminescent agents such as peroxidase enzymes activated with peroxidase substrates capable of luminescence. Photosensitive metal compounds may be present in a light-sensitive matrix or emulsion containing photosensitizable metal compounds, which form metal grains, particles or crystals upon exposure to a developer solution. Developer solutions are formulated to substantially allow living cells to remain viable after exposure to the developing solution. Our methods are useful in flow cytometry and fluorescence activated cell sorting (FACS) methods, microdissection methods, and for attracting, sedimenting, depositing, layering, attaching, adhering, bonding, binding, trapping or suspending cells on surfaces.

대표청구항 ▼

What is claimed is: 1. A method for physically separating cells, the method comprising: providing a liquid comprising a living cell mixture suspended therein, wherein said living cell mixture comprises target cells and non-target cells; labeling said target cells with a photophoric probe adapted to

What is claimed is: 1. A method for physically separating cells, the method comprising: providing a liquid comprising a living cell mixture suspended therein, wherein said living cell mixture comprises target cells and non-target cells; labeling said target cells with a photophoric probe adapted to label said target cells to the exclusion of said non-target cells to form labeled target cells in said liquid, said photophoric probe capable of being controllably activated to produce localized emission of light in the vicinity of said labeled target cells; providing a light sensitive substrate comprising a photosensitizable metal compound; applying said liquid to said light sensitive substrate such that said labeled target cells are adjacent to said light sensitive substrate; activating said photophoric probe to produce localized emission of light in the vicinity of said labeled target cells, said light photosensitizes a portion of said light sensitive substrate to form photosensitized portion of said substrate; developing said photosensitized portions, wherein developing comprises applying to said substrate a developing solution formulated to substantially allow said living cells to remain viable after exposure to said developing solution; forming metal grains from said metal compound whereby said labeled target cells adhere to said metal grains; removing non-target cells of said mixture from the substrate, thereby separating said non-target cells from said labeled target cells adhered to the metal grains. 2. The method according to claim 1, wherein activating said photophoric probe to produce light or to induce the production of light in the vicinity of said labeled target cell comprises using a photoactivation method, a chemical activation method, a thermal activation method or an electrical activation method. 3. The method according to claim 1, wherein said step of activating comprises inducing said photophoric probe to participate in a light producing chemical reaction. 4. The method according to claim 1, wherein said photosensitizable metal compound comprises a photosensitizable metal salt. 5. The method according to claim 4 wherein said photosensitizable metal salt comprises a silver halide. 6. The method according to claim 1, further comprising the step of detaching from said substrate at least one of said labeled target cells adhered to said substrate. 7. The method according to claim 6, further comprising the step of collecting said detached at least one target cell from said substrate. 8. The method according to claim 7, further comprising treating the collected, detached target cell with a metal dissolving solution for dissolving metal grains attached to said collected, detached target cell. 9. The method according to claim 8, wherein said photosensitizable metal compound comprises a photosensitizable silver compound, said metal grains are silver metal grains, and wherein said metal dissolving solution is a solution comprising K3Fe(CN)6 and NH4SCN. 10. The method according to claim 6, wherein said step of detaching comprises applying a detaching agent to said substrate. 11. The method according to claim 10, wherein said detaching agent is n-hexane, or a solution comprising an enzyme capable of detaching the target cell attached to said substrate. 12. The method according to claim 11, wherein said enzyme is a proteolytic enzyme. 13. The method according to claim 12, wherein said proteolytic enzyme is pepsin, trypsin, papain, or combination thereof. 14. The method according to claim 10, wherein said substrate comprises a polymerized calcium alginate matrix and said detaching agent comprises a solution containing a calcium sequestering agent. 15. The method according to claim 14, wherein said calcium sequestering agent comprises sodium citrate, EDTA, or EDTA salts. 16. The method according to claim 1, wherein said target cells to be separated or sorted, are eukaryotic cells, prokaryotic cells, mammalian cells, non-mammalian cells, viable cells, pathogenic organisms, non-pathogenic organisms, bacterial cells, viruses, nanobacteria, unicellular organisms, or multicellular organisms. 17. A method for physically separating cells, the method comprising: providing a liquid comprising a living cell mixture suspended therein, wherein said living cell mixture comprises target cells and non-target cells; labeling said target cells with a photophoric probe adapted to label said target cells to the exclusion of said non-target cells to form labeled target cells in said liquid, said photophoric probe capable of being controllably activated to produce localized emission of light in the vicinity of said selectively labeled target cells; providing a light sensitive substrate, said substrate comprising a photosensitizable metal compound; applying said liquid to said light sensitive substrate such that said labeled target cells are adjacent to, or in contact with, the surface of said light sensitive substrate; activating said photophoric probe to produce localized emission of light in the vicinity of said labeled target cells, said light photosensitizes portions of said light sensitive substrate to form photosensitized portions of said substrate; developing said photosensitized portions, wherein developing comprises applying to said substrate a developing solution formulated to substantially allow said living cells to remain viable after exposure to said developing solution; forming metal grains in said photosensitized portions from said metal compound whereby said labeled target cells adhere to said metal grains; separating said non-target cells from said target cells adhered to the metal grains by removing non-target cells from the substrate. 18. The method according to claim 17, wherein said step of activating comprises using a photoactivation method, a chemical activation method, a thermal activation method or an electrical activation method. 19. The method according to claim 17, wherein said step of activating comprises inducing said photophoric probe to participate in a light-producing chemical reaction. 20. The method according to claim 17, wherein said photosensitizable metal compound comprises a silver halide. 21. The method according to claim 20, wherein said silver halide is silver chloride, silver bromide, silver iodide or a combination thereof. 22. The method according to claim 17, wherein said photophoric probe comprises: a first affinity probe capable of specifically and selectively binding to said target cell or to a second affinity probe bound to said target cell; and a second portion linked to said first affinity probe and capable of being controllably induced to emit light or cause the emission of light in the vicinity of said selectively labeled target cell. 23. The method according to claim 22, wherein said first affinity probe is an antibody, a fragment thereof, a toxin having an affinity for at least a portion of a target cell of said target cells, an oligonucleotide probe, a protein based affinity probe, a glycoprotein based affinity probe, a hapten or molecule having an affinity for at least a portion of said target cell. 24. The method according to claim 22, wherein said second portion of said photophoric probe is a chemiluminescent moiety or agent, a fluorescent moiety or agent, an upconverting moiety, particle or agent, an inorganic two photon upconverting anti-stokes phosphor particle, a two photon upconverting dye, a bioluminescent protein, a bioluminescent molecule, a thermoluminescent moiety, agent or particle or an electroluminescent moiety, agent or particle. 25. The method according to claim 22, wherein said second portion of said photophoric probe comprises an enzyme capable of participating activating or catalyzing a chemiluminescent chemical reaction, resulting in the production of light. 26. The method according to claim 22, wherein said second portion of said photophoric probe comprises aequorin or obelin. 27. The method according to claim 22, wherein said second portion of said photophoric probe comprises an enzyme selected from a peroxidase, a phosphatase, an alkaline-phosphatase, a galactosidase, or a β-glucuronidase. 28. The method according to claim 22, wherein said second portion of said photophoric probe comprises an enzyme capable of catalyzing a chemical reaction for producing a reaction product capable of reacting with a chemical in a chemiluminescent reaction, resulting in the production of light. 29. The method according to claim 17, further comprising treating the detached target cell with a metal dissolving solution for dissolving metal grains attached to said target cell. 30. The method according to claim 29, wherein said photosensitizable metal compound comprises a photosensitizable silver compound, said metal grains are silver metal grains, and wherein said metal dissolving solution is a solution comprising K3(Fe(CN)6) and NH4SCN. 31. The method according to claim 17, further comprising detaching from said substrate a living cell attached to said substrate. 32. The method according to claim 31, further comprising collecting said living cell. 33. The method according to claim 32, further comprising washing said living cell to remove most of said developing solution. 34. The method according to claim 17, further comprising the step of detaching from said substrate at least one of said labeled target cells adhered to said substrate. 35. The method according to claim 34, further comprising collecting the detached target cells. 36. The method according to claim 34, wherein detaching comprises applying a detaching agent to said substrate. 37. The method according to claim 36, wherein said detaching agent is n-hexane or a solution comprising an enzyme capable of detaching the target cell attached to said substrate. 38. The method according to claim 37, wherein said enzyme is a proteolytic enzyme. 39. The method according to claim 38, wherein said proteolytic enzyme is pepsin, trypsin, papain or a combination thereof. 40. The method according to claim 36, wherein said detaching agent is formulated for dissolving portions of said metal grains. 41. The method according to claim 17, wherein said target cells to be separated or sorted are eukaryotic cells, prokaryotic cells, mammalian cells, non-mammalian cells, viable cells, pathogenic organisms, non-pathogenic organisms, bacterial cells, viruses, nanobacteria, unicellular organisms, or multicellular organisms. 42. A method for physically separating cells, the method comprising: providing a liquid containing a mixture of different live cells suspended therein, said mixture comprises target cells and non-target cells; labeling said non-target cells of said mixture with a photophoric probe adapted to label said non-target cells to the exclusion of said target cells to form labeled non-target cells in said mixture, said photophoric probe is capable of being controllably activated to produce localized emission of light in the vicinity of said labeled non-target cells; providing a light sensitive substrate, said substrate comprises at least one photosensitizable metal compound; applying said liquid to said light sensitive substrate such that most labeled non-target cells are adjacent to, or in contact with, the surface of said light sensitive substrate; activating said photophoric probe to produce localized emission of light in the vicinity of said labeled non-target cells, said light photosensitizes portions of said light sensitive substrate to form photosensitized portions; developing said photosensitized portions to form metal grains from said metal compound, wherein developing comprises applying to said substrate a developing solution formulated to substantially allow said living cells to remain viable after exposure to said developing solution, whereby said labeled non-target cells adhere to said metal grains; and separating said target cells from said non-target cells adhered to said metal grains by collecting the liquid from the substrate. 43. The method according to claim 42, wherein said step of activating comprises using a photoactivation method, a chemical activation method, a thermal activation method or an electrical activation method. 44. The method according to claim 42, wherein said step of activating comprises inducing said photophoric probe to participate in a light-producing chemical reaction. 45. The method according to claim 42, wherein said photosensitizable metal compound comprises a silver halide. 46. The method according to claim 45, wherein said silver halide is silver chloride, silver bromide, silver iodide or a combination thereof. 47. The method according to claim 42, wherein said photophoric probe comprises: a first affinity probe capable of specifically and selectively binding to said non-target cell or to a second affinity probe bound to said non-target cell; and a second portion linked to said first affinity probe and capable of being controllably induced to emit light or to cause the emission of light in the vicinity of a non-target cell to which said photophoric probe is bound. 48. The method according to claim 47, wherein said first affinity probe is an antibody or a fragment thereof, a toxin having an affinity for a portion of said non-target cell, an oligonucleotide probe, a protein based affinity probe, a glycoprotein based affinity probe, a hapten or a molecule having an affinity for a portion of said non-target cell. 49. The method according to claim 47, wherein said second portion of said photophoric probe is a chemiluminescent moiety or agent, a fluorescent moiety or agent, an upconverting moiety, particle or agent, an inorganic two photon upconverting anti-stokes phosphor particle, a two photon upconverting dye, a bioluminescent protein, a bioluminescent molecule, a thermoluminescent moiety, agent or particle or an electroluminescent moiety, agent or particle. 50. The method according to claim 47, wherein said second portion of said photophoric probe comprises an enzyme capable of participating, activating or catalyzing a chemiluminescent chemical reaction resulting in the production of light. 51. The method according to claim 47, wherein said second portion of said photophoric probe comprises aequorin or obelin. 52. The method according to claim 47, wherein said second portion of said photophoric probe comprises an enzyme, which is a peroxidase, a phosphatase, an alkaline-phosphatase, a galactosidase or a β-glucuronidase. 53. The method according to claim 47, wherein said second portion of said photophoric probe comprises an enzyme capable of catalyzing a chemical reaction for producing a reaction product capable of reacting with a chemical in a chemiluminescent reaction resulting in the production of light. 54. The method according to claim 42, wherein said step of collecting cells comprises obtaining a mixture of cells having a concentration of said target cells that is higher relative to the concentration of said non-target cells. 55. The method according to claim 42, wherein said target cells are eukaryotic cells, prokaryotic cells, mammalian cells, non-mammalian cells, viable cells, pathogenic organisms, non-pathogenic organisms, bacterial cells, viruses, nanobacteria, unicellular organisms, or multicellular organisms.