Cellulose nanofibrillar bioink for 3d bioprinting for cell culturing, tissue engineering and regenerative medicine applications

Abstract

The present invention relates to biomaterial in the form of dispersion of cellulose nanofibrils with extraordinary shear thinning properties which can be converted into desire 3D shape using 3D Bioprinting technology. In this invention cellulose nanofibril dispersion, is processed through different mechanical, enzymatic and chemical steps to yield dispersion with desired morphological and rheological properties to be used as bioink in 3D Bioprinter. The processes are followed by purification, adjusting of osmolarity of the material and sterilization to yield biomaterial which has cytocompatibility and can be combined with living cells. Cellulose nanofibrils can be produced by microbial process but can also be isolated from plant secondary or primary cell wall, animals such as tunicates, algae and fungi. The present invention describes applications of this novel cellulose nanofibrillar bioink for 3D Bioprinting of tissue and organs with desired architecture.

1 . A method comprising:
 providing one or more nanocellulose dispersion as bioink;   3D bioprinting the bioink and human cells;   producing manufactured tissue from the 3D bioprinting of the bioink and human cells;   subjecting the manufactured tissue to one or more drug-, cosmetic-, or product-related testing protocol; and/or   subjecting the manufactured tissue to one or more disease study as a model.   
     
     
         2 . A method comprising:
 providing one or more nanocellulose dispersion as bioink;   3D bioprinting the bioink and human stem cells;   producing manufactured biomatter from the 3D bioprinting of the bioink and human stem cells:   subjecting the manufactured biomatter to 3D cell culturing and/or stem cell differentiation techniques.   
     
     
         3 . (canceled) 
     
     
         4 . A method comprising:
 preparing a cellulose-based bioink by:
 processing cellulose nanofibrillar material using mechanical, enzymatic and/or chemical processing steps to yield a cellulose nanofibrillar dispersion; 
 purifying the cellulose nanofibrillar dispersion; and 
 sterilizing the cellulose nanofibrillar dispersion to yield the cellulose-based bioink. 
   
     
     
         5 . The method of  claim 4 , further comprising adjusting osmolarity of the cellulose nanofibrillar dispersion. 
     
     
         6 . The method of  claim 4 , wherein the cellulose nanofibrillar material comprises cellulose from one or more of a plant primary cell wall, a plant secondary cell wall, algae, bacteria, fungi, or animals. 
     
     
         7 . The method of  claim 6 , wherein the cellulose nanofibrillar material is derived from tunicates. 
     
     
         8 - 11 . (canceled) 
     
     
         12 . The method of  claim 4 , further comprising: constructing 3D bioprinted matter from the cellulose-based bioink and, with or without cells, wherein the 3D bioprinted matter is one or more of scaffold(s), living tissue(s) and/or organ(s). 
     
     
         13 . The method of  claim 12 , further comprising adjusting osmolarity of the cellulose nanofibrillar dispersion. 
     
     
         14 . The method of  claim 12 , wherein the cellulose nanofibrillar material comprises cellulose from one or more of a plant primary cell wall, a plant secondary cell wall, algae, fungi, or animals. 
     
     
         15 . The method of  claim 14 , wherein the cellulose nanofibrillar material comprises tunicates. 
     
     
         16 - 48 . (canceled) 
     
     
         49 . The method of  claim 1 , wherein the nanocellulose is sourced from bacteria. 
     
     
         50 . The method of  claim 49 , wherein the bacteria is chosen from one or more of the  Aerobacter, Acetobacter, Acromobacter, Agrobacterium, Alacaligenes, Azotobacter, Pseudomonas, Rhizobium , and/or  Sarcina.    
     
     
         51 . The method of  claim 50 , wherein the bacteria is chosen from one or more of  Gluconacetobacter xylinus, Acetobacter xylinum, Lactobacillus mali, Agrobacterium tumefaciens, Rhizobium leguminosarum bv. trifolii, Sarcina ventriculi, enterobacteriaceae Salmonella  spp.,  Escherichia coli, Klebsiella pneu - moniae  and species of cyanobacteria. 
     
     
         52 . The method of  claim 6 , wherein the bacteria is chosen from one or more of the  Aerobacter, Acetobacter, Acromobacter, Agrobacterium, Alacaligenes, Azotobacter, Pseudomonas, Rhizobium , and/or  Sarcina.    
     
     
         53 . The method of  claim 52 , wherein the bacteria is chosen from one or more of  Gluconacetobacter xylinus, Acetobacter xylinum, Lactobacillus mali, Agrobacterium tumefaciens, Rhizobium leguminosarum by. trifolii, Sarcina ventriculi, enterobacteriaceae Salmonella  spp.,  Escherichia coli, Klebsiella pneu - moniae  and species of cyanobacteria. 
     
     
         54 - 57 . (canceled)