Methods and means for influencing intercellular communication and intercellular organelle transport

Abstract

The invention relates to a method for the investigation of intercellular communication and intercellular transport, wherein after singularisation cells are investigated for membrane tubes which contain F-actin and myosin, have a diameter of 50 to 400 nm, as a rule are up to 50 micrometers long or in same cases longer, and which span between the cells. The invention further relates to a method wherein the organelle transport between the cells is investigated.

1 . A method for cell analysis and investigating communication and transport between mammalian cells, comprising the steps of
 (i) singularizing cells in a culture medium;   (ii) spreading and plating singularized cells in a monolayer onto a substrate;   (iii) incubating the singularized cells for a predetermined period in a culture medium;   (iv) staining the singularized cells with a fluorescent or luminescent dye, immunofluorescene or other detectable microscopic stain to obtain stained plasma membranes for 3-D cell microscopy, and   (v) microscopically examining the singularized cells for de novo formed membrane tubes which contain F-actin and myosin, have a diameter of 50 to 400 nanometers and, are up to 50 micrometers long, and   (vi) determining the number of membrane tubes which span between the singularized mammalian cells.   
     
     
         2 . The method according to  claim 1 , wherein a test substance is present in the step of incubating singularized mammalian cells in a culture medium. 
     
     
         3 . The method according to  claim 1 , wherein the culture medium comprises a medicament or therapeutic substance. 
     
     
         4 . The method according to  claim 1 , wherein the microscopically examining is effected with a microscope system which allows the observation of different microscopic planes in z-axis. 
     
     
         5 . The method according to  claim 4 , wherein the microscopic system includes a microscope, a Z-stepper and an associated controller. 
     
     
         6 . The method according to  claim 3 , wherein the medicament or therapeutic substance is for the treatment of tumors, of high blood pressure, of viral, bacterial or parasitic infection diseases, disorders of the metabolism, disorders of the nervous system, the psyche or the mind, or of the cholesterol level. 
     
     
         7 . The method according to  claim 2 , wherein the test substance is for use in gene therapy, for cell targeting or in pharmacology. 
     
     
         8 . The method according to  claim 1 , further comprising microscopically monitoring organelle transport between the singularized mammalian cells. 
     
     
         9 . The method according to  claim 8 , wherein the culture medium comprises a chemical compound or suspected pharmaceutically effective substances to be tested for activity in affecting organelle transport 
     
     
         10 . The method according to  claim 8 , further comprising adding to or expressing in a first population of cells one or more substances, wherein the substance is endocytosed by the cells of the first population or expressed constitutively or transiently by the cells of the first population within a first time period; washing said first population of cells;
 mixing the first number of cells with a second population of cells, so that within a second time period intercellular membrane tubes are formed between the cells of the first and second populations.   
     
     
         11 . The method according to  claim 10 , wherein the one or more endocytable or expressed substances are selected from one or more of the group consisting of dyes, fluorescence dyes, LYSOTRACKER radioactive marker substances, luminescence dyes, fluorescing proteins, luminescing proteins, fluorescing peptides, luminescing peptides, proteins coupled with a marker substances, and peptides coupled with a marker substance. 
     
     
         12 . The method according to  claim 10 , wherein the substance is constitutively or transiently expressed in an organelle. 
     
     
         13 . The method according to  claim 1 , wherein a physical device is present in the step of incubating singularized cells for a predetermined period in a culture medium. 
     
     
         14 . The method according to  claim 13 , wherein the physical device is a physical therapeutic device. 
     
     
         15 . The method according to  claim 1 , wherein in the step of incubating singularized cells for a predetermined period in a culture medium, the culture medium is exposed to an energy form to be tested for activity in affecting membrane tube formation, wherein the energy form is selected from the group consisting of sound, vibration, heat, hydromechanical energy, and electromagnetic waves. 
     
     
         16 . The method according to  claim 8 , wherein in the step of incubating singularized cells for a predetermined period in a culture medium, the culture medium is exposed to an energy form to be tested for activity in affecting organelle transport, wherein the energy form is selected from the group consisting of sound, vibration, heat, hydromechanical energy, and electromagnetic waves.