Methods and combination comprising eukaryotic cells and recombinant spider silk protein

Abstract

A method and a combination for the cultivation of eukaryotic cells are provided, as well as a method for preparation of eukaryotic cells. The methods comprise providing a sample of eukaryotic cells to be cultured, applying said sample to a cell scaffold material; and maintaining said cell scaffold material having cells applied thereto under conditions suitable for cell culture. The combination comprises eukaryotic cells and a cell scaffold material. The cell scaffold material comprises a polymer of a spider silk protein.

1 . A method for the cultivation of eukaryotic cells, comprising
 applying a sample of eukaryotic cells to be cultured to a cell scaffold material; and   maintaining said cell scaffold material having cells applied thereto under conditions suitable for cell culture;   
       wherein
 said cell scaffold material comprises a polymer of a spider silk protein consisting of from 140 to 600 amino acid residues and comprising
 a repetitive fragment of from 70 to 300 amino acid residues derived from the repetitive fragment of a spider silk protein; 
 a C-terminal fragment of from 70 to 120 amino acid residues derived from the C-terminal fragment of a spider silk protein; and 
 
 optionally
 an N-terminal fragment of from 100 to 160 amino acid residues derived from the N-terminal fragment of a spider silk protein. 
 
 
     
     
         2 . A method for the preparation of eukaryotic cells, comprising:
 applying a sample of eukaryotic cells to a cell scaffold material;   maintaining said cell scaffold material having cells applied thereto under conditions suitable for cell culture; and   preparing a sample of cells from said cell scaffold material;   
       wherein
 said cell scaffold material comprises a polymer of a spider silk protein consisting of from 140 to 600 amino acid residues and comprising
 a repetitive fragment of from 70 to 300 amino acid residues derived from the repetitive fragment of a spider silk protein; 
 a C-terminal fragment of from 70 to 120 amino acid residues derived from the C-terminal fragment of a spider silk protein; and 
 
 optionally
 an N-terminal fragment of from 100 to 160 amino acid residues derived from the N-terminal fragment of a spider silk protein. 
 
 
     
     
         3 . The method according to  claim 1 , wherein said eukaryotic cells are mammalian cells. 
     
     
         4 . The method according to  claim 1 , wherein said eukaryotic cells are mammalian cells selected from the group consisting of stem cells and cells from islets of Langerhans. 
     
     
         5 . The method according to  claim 1 , wherein said spider silk protein is selected from the group of proteins defined by the formulas REP-CT and NT-REP-CT, wherein
 NT is a protein fragment having from 100 to 160 amino acid residues, which fragment is a N-terminal fragment derived from a spider silk protein;   REP is a protein fragment having from 70 to 300 amino acid residues, wherein said fragment is selected from the group consisting of L(AG) n L, L(AG) n AL, L(GA) n L, and L(GA) n GL, wherein
 n is an integer from 2 to 10; 
 each individual A segment is an amino acid sequence of from 8 to 18 amino acid residues, wherein from 0 to 3 of the amino acid residues are not Ala, and the remaining amino acid residues are Ala; 
 each individual G segment is an amino acid sequence of from 12 to 30 amino acid residues, wherein at least 40% of the amino acid residues are Gly; and 
 each individual L segment is a linker amino acid sequence of from 0 to 20 amino acid residues; and 
   CT is a protein fragment having from 70 to 120 amino acid residues, which fragment is a C-terminal fragment derived from a spider silk protein.   
     
     
         6 . The method according to  claim 1 , wherein said spider silk protein comprises a cell-binding motif. 
     
     
         7 . The method according to  claim 6 , wherein said cell-binding motif is an oligopeptide coupled to a remainder of the spider silk protein via at least one peptide bond. 
     
     
         8 . The method according to  claim 7 , wherein said oligopeptide is coupled to the N-terminal of the remainder of the spider silk protein. 
     
     
         9 . The method according to  claim 7 , wherein said oligopeptide comprises an amino acid sequence selected from the group consisting of RGD, RGE, IKVAV (SEQ ID NO:23), YIGSR (SEQ ID NO:24), EPDIM (SEQ ID NO:25) and NKDIL (SEQ ID NO:26). 
     
     
         10 . The method according to  claim 1 , wherein said cell scaffold material is in a physical form selected from the group consisting of film, foam, fiber and fiber-mesh. 
     
     
         11 . The method according to  claim 2 , wherein said eukaryotic cells are mammalian cells. 
     
     
         12 . The method according to  claim 2 , wherein said eukaryotic cells are mammalian cells selected from the group consisting of stem cells and cells from islets of Langerhans. 
     
     
         13 . The method according to  claim 2 , wherein said spider silk protein is selected from the group of proteins defined by the formulas REP-CT and NT-REP-CT, wherein
 NT is a protein fragment having from 100 to 160 amino acid residues, which fragment is a N-terminal fragment derived from a spider silk protein;   REP is a protein fragment having from 70 to 300 amino acid residues, wherein said fragment is selected from the group consisting of L(AG) n L, L(AG) n AL, L(GA) n L, and L(GA) n GL, wherein
 n is an integer from 2 to 10; 
 each individual A segment is an amino acid sequence of from 8 to 18 amino acid residues, wherein from 0 to 3 of the amino acid residues are not Ala, and the remaining amino acid residues are Ala; 
 each individual G segment is an amino acid sequence of from 12 to 30 amino acid residues, wherein at least 40% of the amino acid residues are Gly; and 
 each individual L segment is a linker amino acid sequence of from 0 to 20 amino acid residues; and 
   CT is a protein fragment having from 70 to 120 amino acid residues, which fragment is a C-terminal fragment derived from a spider silk protein.   
     
     
         14 . The method according to  claim 2 , wherein said spider silk protein comprises a cell-binding motif. 
     
     
         15 . The method according to  claim 14 , wherein said cell-binding motif is an oligopeptide coupled to a remainder of the spider silk protein via at least one peptide bond. 
     
     
         16 . The method according to  claim 15 , wherein said oligopeptide is coupled to the N-terminal of the remainder of the spider silk protein. 
     
     
         17 . The method according to  claim 15 , wherein said oligopeptide comprises an amino acid sequence selected from the group consisting of RGD, RGE, IKVAV (SEQ ID NO:23), YIGSR (SEQ ID NO:24), EPDIM (SEQ ID NO:25) and NKDIL (SEQ ID NO:26). 
     
     
         18 . The method according to  claim 2 , wherein said cell scaffold material is in a physical form selected from the group consisting of film, foam, fiber and fiber-mesh.