Methods for Generation of Cell-derived Microfilament Network

Abstract

This invention provides for a network of cell-derived microfilaments. Also provided are methods of producing a network of microfilaments via culturing cells in a matrix support and cell culture medium wherein the cells proliferate and form aggregated cell masses, which produce microfilaments external to and surrounding the cell masses, and wherein the extracellular microfilaments connect and form a continuous extracellular microfilament network, and methods for treating a medical condition as well as facilitating wound repair and tissue regeneration comprising applying the microfilament network to an area in need of treatment.

1 .- 30 . (canceled) 
     
     
         31 . A method of producing a network of microfilaments comprising the steps of:
 culturing cells in a matrix support and cell culture medium wherein the cells proliferate and form aggregated cell masses, and wherein the cell masses produce microfilaments external to and surrounding the cell masses, and wherein the extracellular microfilaments connect and form a continuous extracellular microfilament network, and   removing nuclei of the cells and/or the cells from the network.   
     
     
         32 . The method of  claim 31  wherein the cell masses form on top of the matrix support. 
     
     
         33 .- 58 . (canceled) 
     
     
         59 . A method for treating a medical condition comprising applying the microfilament network comprising cell-derived microfilaments interconnected in a continuous lattice or mesh structure between a plurality of microfilament source regions to an area in need of treatment. 
     
     
         60 . The method of  claim 59  wherein the microfilament network is applied with the matrix. 
     
     
         61 . The method of  claim 59  wherein the microfilament network is applied without the matrix. 
     
     
         62 . The method of  claim 59  wherein the medical condition is a wound or an injured tissue. 
     
     
         63 . The method of  claim 59  wherein the microfilament network prevents infection of the wound or the injured tissue, and facilitates wound healing and tissue regeneration. 
     
     
         64 . The method of  claim 59 , wherein the medical condition is a burn. 
     
     
         65 . The method of  claim 59  including enhancing or promoting re-epithelialization of damaged skin. 
     
     
         66 . The method of  claim 59 , further including administering therapeutic drugs prior to, concurrent with or after applying the microfilament network to the area in need of treatment. 
     
     
         67 . A method of producing a continuous network of cell-derived microfilaments in vitro comprising:
 culturing a plurality of cell clusters on a surface of a matrix substrate under conditions where the cell clusters produce microfilaments external to and surrounding the cell clusters, and wherein the extracellular microfilaments connect and form a continuous extracellular network of microfilaments between the cell clusters on the surface of the matrix substrate.   
     
     
         68 . The method of  claim 67  wherein the cell clusters are spaced apart by an average distance of between about 1 microns and about 1000 microns. 
     
     
         69 . The method of  claim 67  wherein the microfilaments connecting the cell clusters have an average length of between about 10 microns and about 100 microns. 
     
     
         70 . The method of  claim 67  wherein the continuous extracellular microfilament network is a multilayered lattice. 
     
     
         71 . The method of  claim 67  wherein the microfilaments are branched. 
     
     
         72 . The method of  claim 67  wherein the continuous extracellular microfilament network is a multilayered lattice including long microfilaments having a length of between about 10 microns and about 1000 microns and short microfilaments having a length of between about 1 microns and about 10 microns. 
     
     
         73 . The method of  claim 67  wherein the continuous extracellular network of microfilaments has a surface area of between about 0.01 cm 2  and 500 cm 2 . 
     
     
         74 . The method of  claim 67  wherein the continuous extracellular microfilament network is a mesh. 
     
     
         75 . The method of  claim 67  wherein the continuous extracellular microfilament network is porous. 
     
     
         76 . The method of  claim 67  wherein cells are implanted on the surface of the matrix substrate and wherein the cells migrate and aggregate into preclusters and proliferate to form the cell clusters. 
     
     
         77 . The method of  claim 67  further including removing cell nuclei or the cell clusters from the continuous extracellular microfilament network. 
     
     
         78 . The method of  claim 67  wherein the matrix substrate inhibits attachment of the cells. 
     
     
         79 . The method of  claim 67  further including separating the matrix substrate from the continuous extracellular microfilament network. 
     
     
         80 . A method for facilitating wound repair and/or tissue regeneration comprising applying the microfilament network comprising cell-derived microfilaments interconnected in a continuous lattice or mesh structure between a plurality of microfilament source regions to a site in need thereof. 
     
     
         81 . The method of  claim 80  wherein the microfilament network is applied with the matrix. 
     
     
         82 . The method of  claim 80  wherein the microfilament network is applied without the matrix. 
     
     
         83 . The method of  claim 80  wherein the microfilament network form square foot (ft 2 )-scale ultra-large microfilament lattices (UMLs). 
     
     
         84 . The method of  claim 83  wherein the UMLs construct an environment for cell migration. 
     
     
         85 . The method of  claim 80  wherein the microfilament network are multilayered and three dimensional (3D). 
     
     
         86 . The method of  claim 80  including removing cell masses and producing acellular UMLs (AUMLs). 
     
     
         87 . The method of  claim 86  wherein the AUMLs facilitate wound repair. 
     
     
         88 . The method of  claim 86  including applying the AUMLs to a wounded site. 
     
     
         89 . The method of  claim 88  wherein applying the AUMLs allows keratinocytes to engender large tunnels in the reepithelialized epidermis. 
     
     
         90 . The method of  claim 89  wherein the large tunnels provide pathways for cells and nutrients to the site of wound repair. 
     
     
         91 . The method of  claim 80  wherein the microfilament network prevents infection of the wounded site. 
     
     
         92 . The method of  claim 80  including enhancing or promoting re-epithelialization of wounded skin. 
     
     
         93 . The method of  claim 80  wherein the wound is second degree thermal burn wounds. 
     
     
         94 . The method of  claim 80 , further including administering therapeutic drugs prior to, concurrent with or after applying the microfilament network to the area in need of treatment.