Methods for modeling infectious disease and chemosensitivity in cultured cells and tissues

Abstract

The present invention provides methods for utilizing a form of optimized suspension culture to examine the infectivity of pathogenic organisms and agents in human cells and tissues. Also provided are methods using a rotating wall vessel to predict Chemosensitivity of cells and tissues to toxins and chemotherapeutic agents. These culture conditions potentiate spatial colocalization and three-dimensional assembly of individual cells into large aggregates which more closely resemble the in vivo tissue equivalent. In this environment, dissociated cells can assemble and differentiate into macroscopic tissue aggregates several millimeters in size. These culture conditions allow for better cellular differentiation and formation of three-dimensional cellular aggregates, more efficient cell-to-cell interactions, the in in vivo-like exchange of growth factors and greater molecular scaffolding facilitating mechanical stability for cells. The suspension culture system offers a new approach for studying microbial infectivity from the perspective of the host-pathogen interaction and also for analyzing chemosensitivity to toxins and chemotherapeutic agents.

1 .- 25 . (canceled) 
   
   
       26 . A method of studying infectivity of a pathogen in tissues comprising the steps of:
 isolating cells from a host tissue sample;   placing said isolated host cells into a bioreactor comprising culture medium to form a cell culture;   applying sedimental shear stress to the cells in the cell culture to form a three-dimensional tissue mass;   seeding the formed tissue mass in a tissue culture vessel;   introducing an infectious pathogen into said three-dimensional mass; and   assaying the infectivity of said infectious pathogen, wherein said assaying comprises the steps of:
 obtaining a measurement of at least one of the following parameters after introducing said infectious pathogen:
 adherence of said infectious pathogen to cultured cells; 
 cytokine expression in cultured cells; 
 prostaglandin synthesis in cultured cells; 
 mRNA or protein expression in cultured cells; 
 invasion of said pathogen into cultured cells; 
 death or damage of cultured cells; or 
 tissue pathology of aggregates of cultured cells; and 
 
   comparing the measurement to a control value of the measured parameter, wherein the control value corresponds to a measurement of the same parameter for a cell culture into which said infectious pathogen has not been introduced.   
   
   
       27 . The method of  claim 26 , optionally comprising use of a culture matrix in said bioreactor that facilitates growth of said host cells. 
   
   
       28 . The method of  claim 26 , wherein the bioreactor is a rotating wall vessel. 
   
   
       29 . The method of  claim 26 , wherein said isolated host cells are epithelial cells. 
   
   
       30 . The method of  claim 29 , wherein said epithelial cells are human cells originating from lung, kidney, liver, intestine or bladder. 
   
   
       31 . The method of  claim 27 , wherein said culture matrix consists of microbeads or microcarriers. 
   
   
       32 . The method of  claim 26 , wherein said infectious pathogen is selected from the group consisting of viruses, bacteria, protozoa, parasites and fungi. 
   
   
       33 . The method of  claim 32  wherein said infectious pathogen is  Salmonella typhimurium.    
   
   
       34 . The method of  claim 26 , wherein said culture medium comprises fetal bovine serum and a tri-sugar based medium comprising fructose, galactose and lactose. 
   
   
       35 . The method of  claim 31 , wherein said microbeads are collagen-coated microbeads. 
   
   
       36 . A method of studying the infectivity of a pathogen in tissues comprising the steps of:
 isolating cells from a sample of intestinal epithelial tissue;   placing said intestinal epithelial cells into a bioreactor comprising culture medium to form a cell culture;   applying sedimental shear stress to the cells in the cell culture to form a three dimensional tissue mass;   seeding the formed tissue mass in a tissue culture vessel;   introducing an infectious pathogen to the formed tissue mass;   assaying the infectivity of said infectious pathogen, wherein said assaying comprises the steps of:
 obtaining a measurement of at least one of the following parameters after introducing said infectious pathogen:
 adherence of said infectious pathogen to cultured cells; 
 cytokine expression in cultured cells; 
 prostaglandin synthesis in cultured cells; 
 mRNA or protein expression in cultured cells; 
 invasion of said pathogen into cultured cells; 
 death or damage of cultured cells; or 
 tissue pathology of aggregates of cultured cells; and 
 
 comparing the measurement to a control value of the measured parameter, wherein the control value corresponds to a measurement of the same parameter for a cell culture into which said infectious pathogen has not been introduced. 
   
   
   
       37 . The method of  claim 36 , wherein said infectious pathogen is  Salmonella typhimurium.    
   
   
       38 . A method of measuring the chemosensitivity of tissues to a toxic material comprising:
 isolating cells from a host tissue sample;   placing said isolated host cells into a bioreactor comprising culture medium to form a cell culture;   applying sedimental shear stress to the cells in the cell culture to form a three-dimensional tissue mass;   seeding the formed tissue mass in a tissue culture vessel;   introducing a toxic material into said three-dimensional tissue mass; and   assaying the chemosensitivity of said toxic material by obtaining a measurement of cell death or damage among cultured cells after introduction of said toxic material; and   comparing the measurement to a control value of cell death or damage for a cell culture into which said toxic material has not been introduced.   
   
   
       39 . The method of  claim 38 , optionally comprising use of a culture matrix that facilitates growth of said host cells. 
   
   
       40 . The method of  claim 38 , wherein said isolated host cells are epithelial cells. 
   
   
       41 . The method of  claim 40 , wherein said epithelial cells are human renal cells. 
   
   
       42 . The method of  claim 39 , wherein said culture matrix consists of microbeads or microcarriers. 
   
   
       43 . The method of  claim 42 , wherein said microbeads are collagen-coated microbeads. 
   
   
       44 . The method of  claim 38 , wherein said toxic material is a chemotherapeutic material. 
   
   
       45 . The method of  claim 44 , wherein said chemotherapeutic material is an antibiotic. 
   
   
       46 . The method of  claim 45 , wherein said antibiotic is gentamicin. 
   
   
       47 . The method of  claim 38 , wherein said culture medium comprises fetal bovine serum and DMEM/F12. 
   
   
       48 . A method of measuring the chemosensitivity of tissues to a toxic material comprising:
 isolating cells from a sample of human renal epithelial tissue;   placing said isolated human epithelial cells into a bioreactor comprising culture medium to form a cell culture;   applying sedimental shear stress to the cells in the cell culture to form a three-dimensional tissue mass;   seeding the formed tissue mass in a tissue culture vessel;   contacting the three dimensional tissue mass with a toxic material; and   assaying the chemosensitivity of said toxic material by obtaining a measurement of cell death or damage among cultured cells after introduction of said toxic material; and   comparing the measurement to a control value of cell death or damage for a cell culture into which said toxic material has not been introduced.   
   
   
       49 . The method of  claim 48 , wherein said toxic material is a chemotherapeutic material. 
   
   
       50 . The method of  claim 49 , wherein said chemotherapeutic material is an antibiotic.