Ferromagnetic Cell and Tissue Culture Microcarriers

Abstract

A porous, collagen coated, ferromagnetic cell culture microcarrier, which is suitable for in vitro cell and tissue culture and which facilitates 3D multicellular construct generation. Also provided is a method for creating batches of microcarriers which have inserted within them magnetite (Fe3O4) in the presence of collagen, thus creating a microcarrier which becomes magnetic in nature when placed in a the presence of a magnetic field and which facilitates cellular adherence (via the collagen coating) for 3D construct development.

1 . A method of developing a three-dimensional cell culture model, comprising the steps of; 
 providing a plurality of microcarriers;    providing an upper and lower graphite plate;    providing an adjustable platform, whereby the upper and lower plate are adjustably spaced;    adhering at least one cell to the microcarriers in a gas-permeable tissue culture bag;    placing the tissue culture bag between the upper graphite plate and the lower graphite plate; and    placing a magnet above the upper graphite plate whereby the growing cells are levitated by adjustment of the platform to the appropriate height and are held in suspension via natural magnetic forces stabilized by the graphite plates, thereby allowing the study of three-dimensional cell growth in suspension in a constant and nonrandomized environment.    
   
   
       2 . The method of  claim 1  wherein the microcarriers are ferromagnetic microcarriers.  
   
   
       3 . The method of  claim 1  wherein the upper and lower graphite plates are pyrolyric graphite plates.  
   
   
       4 . The method of  claim 1  wherein the adjustable platform is placed in a standard incubator.  
   
   
       5 . A microcarrier bead having a supporting surface for the attachment of cells, the microcarrier bead further comprising; 
 at least one magnetically charged molecule; and    a cellular matrix material.    
   
   
       6 . The microcarrier of  claim 5  wherein the magnetically charged molecule is magnetite (Fe 3 O 4 ).  
   
   
       7 . The microcarrier of  claim 5  wherein the cellular matrix material is Type I solubilized collagen.  
   
   
       8 . The microcarrier of  claim 5  wherein the support material is constructed from porous gelatin.  
   
   
       9 . A method of manufacturing a gelatin microcarrier bead having a supporting surface for the attachment of cells, comprising the steps of; 
 swelling a porous gelatin microcarrier in culture media;    sterilizing the swelled microcarrier;    suspending the sterilized microcarriers in an acidic solution;    rotating the solution for a first predetermined time, at a first predetermined temperature;    rotating the solution for a second predetermined time, at a second predetermined temperature; and    stabilizing the loaded microcarriers prior to use.    
   
   
       10 . The method of  claim 9  wherein the gelatin microcarriers range in size from about 100-400 μM.  
   
   
       11 . The method of  claim 9  wherein the culture media is void of calcium and magnesium.  
   
   
       12 . The method of  claim 9  wherein the ratio of microcarriers to culture media is about 0.5 g beads/25 ml media.  
   
   
       13 . The method of  claim 9  wherein the microcarriers are sterilized in an autoclave.  
   
   
       14 . The method of  claim 9  wherein the acidic solution further comprises 1 mg Type I solubilized collagen and 25 mg sterile fine granular magnetite (Fe 3 O 4 ).  
   
   
       15 . The method of  claim 14  wherein the acidic solution has a final volume of about 2 ml.  
   
   
       16 . The method of  claim 9  wherein the first predetermined time is about 4 to 8 hours.  
   
   
       17 . The method of  claim 9  wherein the first predetermined temperature is about room temperature.  
   
   
       18 . The method of  claim 9  wherein the second predetermined time is about 24-48 hours.  
   
   
       19 . The method of  claim 9  wherein the second predetermined temperature is about 37 degrees Celsius.  
   
   
       20 . The method of  claim 9  wherein the step of stabilizing the loaded microcarriers further comprises the steps of: 
 washing the microcarriers to remove any excess collagen and magnetite (Fe 3 O 4 ); and    storing the microcarriers in a protein-containing media at a neutral pH under sterile conditions at 4 degrees Celsius.    
   
   
       21 . A method of manufacturing an alginate microcarrier bead having a supporting surface for the attachment of cells, comprising the steps of; 
 providing a solution comprising alginate and culture media lacking calcium and magnesium;    adding sterile fine granular magnetite (Fe 3 O 4 ) to the solution;    expressing the combined solution in droplet form into a solution of calcium chloride wherein the microcarrier bead is formed in about 1-2 hours;    washing the microcarrier beads with culture media; and    coating the microcarrier beads with a collagen solution.    
   
   
       22 . The method of  claim 21  wherein the solution comprising alginate and culture media lacking calcium and magnesium contains about 2% alginate (about 0.5 g alginate per 25 ml).  
   
   
       23 . The method of  claim 21  wherein the amount of sterile fine granular magnetite (Fe 3 O 4 ) is about 150 mg.  
   
   
       24 . The method of  claim 21  wherein the solution of calcium chloride is about 25 Nm calcium chloride.  
   
   
       25 . The method of  claim 21  wherein the collagen solution is about 0.3 mg solubilized Type I collagen.