Cultured tissue and bioreactor systems and methods for production thereof

Abstract

The present disclosure relates to cultured tissue, methods for production of the cultured tissue, and a bioreactor system for production of the cultured tissue. In some embodiments, the production of the cultured tissue may involve, at a first bioreactor, feeding a fiber scaffold into a chamber containing culture media, seeding the chamber with precursor cells, and allowing the precursor cells to proliferate and differentiate on a surface of the fiber scaffold. At downstream bioreactors, the production of the cultured tissue may further involve twisting a plurality of the cell-laden fibers to provide a cell-laden yarn, and weaving or knitting the cell-laden yarn into a three-dimensional (3D) structure. In some embodiments, the cultured tissue may be whole muscle cultured meat composed of muscle cell-laden fibers and fat cell-laden fibers. The whole muscle cultured meat may have a structural organization and hierarchy that mimics natural skeletal muscle tissue.

What is claimed: 
     
         1 . A system for the production of cultured tissue, comprising:
 a first bioreactor including
 an internal chamber containing culture medium, 
 a fiber inlet for feeding a fiber scaffold into the internal chamber, and 
 a cell inlet for feeding precursor cells into the internal chamber, wherein the precursor cells proliferate and differentiate on a surface of the fiber scaffold in the culture medium to provide a cell-laden fiber composed of cells attached to the fiber scaffold, and 
 an outlet through which the cell-laden fiber emerges from the first bioreactor, wherein the cell-laden fiber is used in the production of the cultured tissue. 
   
     
     
         2 . The system of  claim 1 , further comprising a second bioreactor downstream of the first bioreactor and configured to combine and twist the cell-laden fibers emerging from one or more of the first bioreactors to provide a cell-laden yarn. 
     
     
         3 . The system of  claim 2 , wherein the second bioreactor includes wheels attached to each end of the cell-laden fibers which rotate at a rotation rate to twist the cell-laden fibers. 
     
     
         4 . The system of  claim 2 , further comprising a third bioreactor downstream of the second bioreactor and configured to weave or knit the cell-laden yarn into a three-dimensional (3D) structure. 
     
     
         5 . The system of  claim 2 , further comprising a third bioreactor configured to weave or knit the cell-laden yarn from the second bioreactor into a two-dimensional (2D) sheet. 
     
     
         6 . The system of  claim 5 , wherein the third bioreactor is further configured to roll the 2D sheet into a three-dimensional (3D) structure. 
     
     
         7 . The system of  claim 5 , wherein the third bioreactor is further configured to stack a plurality of the 2D sheets into a three-dimensional (3D) structure. 
     
     
         8 . The system of any of  claims 4  to  7 , wherein the 2D sheet or the 3D structure provides the cultured tissue. 
     
     
         9 . The system of any of  claims 1  to  8 , wherein the cell-laden fiber includes muscle cells, fat cells, or a combination thereof. 
     
     
         10 . The system of any of  claims 1  to  9 , wherein the cell-laden fibers of the cultured tissue includes muscle cell-laden fibers, fat cell-laden fibers, or a combination of muscle cell-laden fibers and fat cell-laden fibers. 
     
     
         11 . The system of any of  claims 4  to  10 , wherein the first bioreactor, the second bioreactor, and the third bioreactor are configured to operate automatically and continuously to produce the cultured tissue. 
     
     
         12 . The system of  claim 11 , further comprising one or more computer controllers in communication with the first bioreactor, the second bioreactor, and the third bioreactor for automating the operation of the first bioreactor, the second bioreactor, and the third bioreactor. 
     
     
         13 . The system of  claim 12 , wherein the one or more computer controllers are configured to control one or more of a time frame for proliferation and differentiation of the precursor cells at the first bioreactor, a degree of twisting of the cell-laden fibers at the second bioreactor, a rotation rate of the wheels of the second bioreactor, a composition of the cultured tissue, a size of the cultured tissue, a cell density of the cultured tissue, a packing density of the cultured tissue, and a ratio of muscle cell-laden fibers and fat cell-laden fibers in the cultured tissue. 
     
     
         14 . The system of any preceding claim, wherein the first bioreactor further comprises a first spool run by a motor which feeds the fiber scaffold into the fiber inlet. 
     
     
         15 . The system of  claim 14 , wherein the first bioreactor further comprises a cover over the first spool. 
     
     
         16 . The system of any preceding claim, wherein the first bioreactor further comprises inlet ports for replenishing the culture media. 
     
     
         17 . The system of any preceding claim, wherein the first bioreactor further comprises one or more rods in the internal chamber configured to contact the fiber scaffold to keep the fiber scaffold submerged in the culture media. 
     
     
         18 . The system of any preceding claim, further comprising a second spool run by a motor configured to spool the cell-laden fiber emerging from the outlet of the first bioreactor. 
     
     
         19 . The system of any preceding claim, wherein the cells are cultured to at least 75% confluence in the first bioreactor. 
     
     
         20 . The system of any preceding claim, wherein a concentration of growth factors in the culture media decreases from a proximal end to a distal end of the first bioreactor. 
     
     
         21 . The system of any preceding claim, wherein the first bioreactor further comprises a body having one or more translucent sections to allow observation of the internal chamber. 
     
     
         22 . A method for producing of cultured tissue, comprising:
 feeding a fiber scaffold into a chamber containing culture medium;   seeding the chamber with precursor cells;   allowing the precursor cells to proliferate and differentiate on a surface of the fiber scaffold to provide a cell-laden fiber composed of cells adhered to the fiber scaffold;   twisting a plurality of cell-laden fibers to provide a cell-laden yarn; and   weaving or knitting the cell-laden yarn into a three-dimensional (3D) structure to provide the cultured tissue.   
     
     
         23 . The method of  claim 22 , wherein the method is operated automatically and continuously. 
     
     
         24 . The method of  claim 22  or  23 , wherein the method is robotically operated. 
     
     
         25 . The method of any of  claims 22  to  24 , further comprising applying a coating to the fiber scaffold to improve attachment of the cells to the fiber scaffold. 
     
     
         26 . The method of any of  claims 22  to  25 , wherein weaving or knitting the cell-laden yarn comprises weaving or knitting the cell-laden yarn into a two-dimensional (2D) sheet, followed by rolling or folding the 2D sheet into the 3D structure. 
     
     
         27 . The method of any of  claims 22  to  25 , wherein weaving or knitting the cell-laden yarn comprises weaving or knitting the cell-laden yarn into a two-dimensional (2D) sheet, followed by stacking a plurality of the 2D sheets into the 3D structure. 
     
     
         28 . The method of any of  claims 22  to  27 , wherein feeding the fiber scaffold into the chamber, seeding the chamber with precursor cells, and allowing the precursor cells to proliferate and differentiate on the surface of the fiber scaffold is carried out at a first bioreactor. 
     
     
         29 . The method of  claim 28 , wherein twisting the plurality of cell-laden fibers is carried out at a second bioreactor downstream of the first bioreactor. 
     
     
         30 . The method of  claim 29 , wherein weaving or knitting the cell-laden yarn into the 3D structure is carried out at a third bioreactor downstream of the second bioreactor. 
     
     
         31 . The system or the method of any preceding claim, wherein the culture medium is a simultaneous proliferation/differentiation media capable of supporting both cell proliferation and cell differentiation. 
     
     
         32 . The system or method of any preceding claim, wherein the culture medium includes Dulbecco's Modified Eagle's Medium (DMEM), from 0% to 10% fetal bovine serum (FBS), 1% antiobiotic-antimycotic, from 1 nanogram (ng)/milliliter (mL) to 100 ng/mL insulin like growth factor 1 (ILGF-1), and 10 micrograms (μg)/mL insulin. 
     
     
         33 . The system or method of  claim 32 , wherein the culture medium includes DMEM, 10% FBS, 100 ng/mL ILGF1, and 10 μg/mL insulin. 
     
     
         34 . The system or method of any preceding claim, wherein the cells include tetracycline responsive promoters for expression of myogenic or adipogenic genes, and wherein the culture medium includes tetracycline. 
     
     
         35 . Cultured tissue comprising a plurality of cell-laden fibers each comprised of cells attached to a fiber scaffold, wherein the plurality of cell-laden fibers are twisted into a cell-laden yarn, wherein the cell-laden yarn is further woven or knitted into a three-dimensional (3D) shape, and wherein the cultured tissue exhibits a structural organization of the cell-laden fibers that mimics skeletal muscle tissue. 
     
     
         36 . The system, the method, or the cultured tissue of any preceding claim, wherein the cultured tissue is cultured meat for consumption. 
     
     
         37 . The system, the method, or the cultured tissue of any preceding claim, wherein a stiffness of the cultured tissue is about 12 kilopascals. 
     
     
         38 . The system, the method, or the cultured tissue of any preceding claim, wherein the cells are engineered to produce vital nutrients. 
     
     
         39 . The system, the method, or the cultured tissue of any preceding claim, wherein the cultured tissue is composed of muscle cell-laden fibers, fat cell-laden fibers, or a combination of muscle cell-laden fibers and fat cell-laden fibers. 
     
     
         40 . The system, the method, or the cultured tissue of any preceding claim, wherein the cultured tissue is composed of a combination of muscle cell-laden fiber fibers and fat cell-laden fibers. 
     
     
         41 . The system, the method, or the cultured tissue of  claim 40 , wherein the cultured tissue exhibits marbling based on a ratio of the muscle cell-laden fibers and the fat cell-laden fibers in the cultured tissue. 
     
     
         42 . The system, the method, or the cultured tissue of  claim 41 , wherein the marbling of the cultured tissue resembles marbling in skeletal muscle tissue. 
     
     
         43 . The system, the method, or the cultured tissue of any preceding claim, wherein the fiber scaffold is edible. 
     
     
         44 . The system, the method, or the cultured tissue of any preceding claim, wherein the fiber scaffold has a tensile strength that ranges from 3 kilopascals (kPa) to 40 kPa. 
     
     
         45 . The system, the method, or the cultured tissue of any preceding claim, wherein the fiber scaffold is composed of a material selected from the group consisting of collagen, silk, chitosan, wheat gluten, cellulose, zein, starch, soy protein, fungal mycelia, mung bean fiber and combinations thereof. 
     
     
         46 . The system, the method, or the cultured tissue of any preceding claim, wherein the cells are edible. 
     
     
         47 . The system, the method, or the cultured tissue of any preceding claim, wherein the cells are muscle cells, fat cells, or combinations thereof. 
     
     
         48 . The system, the method, or the cultured tissue of any preceding claim, wherein precursor cells or the cells are selected from the group consisting of bovine satellite cells, chicken fibroblasts, quail muscle cells, salmon cells, tuna cells, and combinations thereof. 
     
     
         49 . The system, the method, or the cultured tissue of any preceding claim, wherein the fiber scaffold includes a coating that improves adhesion of the cells to the fiber scaffold. 
     
     
         50 . The system, the method, or the cultured tissue of any preceding claim, wherein the fiber scaffold is coated with a coating composition selected from the group consisting of fibronectin, laminin, vitronectin, collagen, cadherin, elastin, hyaluronic acid, poly-D-lysine, poly-L-ornithine, concanavalin A, soy, and combinations thereof. 
     
     
         51 . The system, the method, or the cultured tissue of any preceding claim, wherein the fiber scaffold is coated with laminin or collagen. 
     
     
         52 . The system, the method, or the cultured tissue of any preceding claim, wherein the fiber scaffold is coated with a non-toxic adhesive chemical. 
     
     
         53 . The system, the method, or the cultured tissue of any preceding claim, wherein the fiber scaffold is includes a coating of extracellular matrix proteins. 
     
     
         54 . The system, the method, or the cultured tissue of any preceding claim, wherein at least 75% of a surface of the fiber scaffold is covered with the cells in the cell-laden fibers. 
     
     
         55 . The system, the method, or the tissue of any preceding claim, wherein a diameter of the cell-laden yarn ranges from 50 micrometers to 100 micrometers. 
     
     
         56 . The system, the method, or the tissue of any preceding claim, wherein a density of the cell-laden yarn is about 1.06 kilogram/liter. 
     
     
         57 . The system, the method, or the tissue of any of  claims 1  to  55 , wherein a density of the cell-laden yarn is about 0.92 kilogram/liter.