Mammalian Lung Spheroids and Lung Spheroid Cells and Uses Thereof

Abstract

This invention relates generally to the discovery of novel mammalian lung spheroids and lung spheroid cells and uses thereof.

What is claimed is: 
     
         1 . A method for preparing therapeutically useful mammalian lung spheroids which comprises (i) culturing mammalian lung tissue explant cells under adherent culture conditions to form a first lung cell outgrowth culture; (ii) culturing the first lung cell outgrowth culture under low-adherence conditions to form lung spheroid clusters; and (iii) recovering the therapeutically useful mammalian lung spheroids. 
     
     
         2 . The method of  claim 1 , wherein the mammalian lung spheroids are human lung spheroids. 
     
     
         3 . The method of  claim 1 , wherein the adherent culture conditions in step (i) are a glycoprotein-coated, a protein-coated, or a proteoglycan-coated surface. 
     
     
         4 . The method of  claim 3  wherein the glycoprotein-coated surface is a collagen-coated surface, a fibronectin-coated surface, or a laminin-coated surface. 
     
     
         5 . The method of  claim 1 , wherein the adherent culture conditions in step (i) are an uncoated plastic surface. 
     
     
         6 . The method of  claim 1 , wherein the low-adherence conditions comprise a bioreactor or a neutrally-charged hydrogel-coated surface. 
     
     
         7 . The method of  claim 1 , wherein the mammalian lung spheroids are (i) positive for antibodies to CCSP, CD105, CD90, and Pro-SPC and (ii) negative for antibodies to CD31, CD34, and CD45. 
     
     
         8 . The method of  claim 1 , wherein the culturing conditions comprise a media that consists essentially of Iscove's Modified Dulbecco's Media (IMDM) and fetal bovine serum (FBS). 
     
     
         9 . A method for preparing therapeutically useful mammalian lung spheroid cells which comprises (i) culturing human lung tissue explant cells under adherent culture conditions to form a first lung cell outgrowth culture; (ii) culturing the first lung cell outgrowth culture under low-adherence conditions to form lung spheroids; and (iii) culturing the lung spheroids under adherent culture conditions so as to form the therapeutically useful mammalian lung spheroid cells. 
     
     
         10 . The method of  claim 9 , wherein the mammalian lung spheroid cells are human lung spheroid cells. 
     
     
         11 . The method of  claim 9 , wherein the adherent culture conditions in step (i) are a glycoprotein-coated, a protein-coated, or a proteoglycan-coated surface. 
     
     
         12 . The method of  claim 11 , wherein the glycoprotein-coated surface is a collagen-coated surface, a fibronectin-coated surface or a laminin-coated surface. 
     
     
         13 . The method of  claim 9 , wherein the adherent culture conditions in step (i) are an uncoated plastic surface. 
     
     
         14 . The method of  claim 9 , wherein the low-adherence conditions comprise a bioreactor or a neutrally-charged hydrogel-coated surface. 
     
     
         15 . The method of  claim 9 , wherein the mammalian lung spheroid cells are (i) positive for antibodies to CCSP, CD105, CD90, and Pro-SPC and (ii) negative for antibodies to CD31, CD34, and CD45. 
     
     
         16 . A method for the treatment of a lung disease in a mammalian patient which comprises providing to the patient a therapeutically effective amount of mammalian lung spheroids wherein the lung spheroids were prepared by (i) culturing a mammalian lung tissue explant under adherent culture conditions to for a first lung cell outgrowth culture; and (ii) culturing the first lung cell outgrowth culture under low-adherence conditions to form lung spheroids. 
     
     
         17 . The method of  claim 16 , wherein the mammalian lung spheroids are human lung spheroids. 
     
     
         18 . (canceled) 
     
     
         19 . The method of  claim 16 , wherein the mammalian patient is a veterinary patient. 
     
     
         20 .- 41 . (canceled) 
     
     
         42 . A pharmaceutical composition comprising mammalian lung spheroids of  claim 1  wherein (a) the lung spheroids have a diameter of about 25 μM to about 500 μM; (b) (i) are negative or slightly positive for c-Kit; (ii) are negative for at least one hematopoietic marker; and (iii) are negative for at least one cardiosphere marker; and (c) a pharmaceutically acceptable carrier. 
     
     
         43 . A pharmaceutical composition comprising mammalian lung spheroid cells of  claim 1  wherein (a) (i) are negative or slightly positive for c-Kit; (ii) are negative for at least one hematopoietic marker; and (iii) are negative for at least one cardiosphere marker; and (b) a pharmaceutically acceptable carrier. 
     
     
         44 . (cance