Method for culturing langerhans islets and islet autotransplantation islet regeneration

Abstract

A method for culturing Langerhans islets to obtain an amount sufficient for transplant and autotransplant is disclosed The islets are cultured in a culture serum (rat/human) medium which is supplemented with radical scavengers, growth factors, a matrix material, nerve growth factor, cell migrating/scattering factors and anti-integrin β1 antibody at proper the time during the culturing process. The medium is supplemented with radical scavengers and growth factors for the first time and then further supplemented with matrix material, radical scavengers, nerve growth factor and the growth factors around 12-24 hours after culturing. Thereafter, the medium is supplemented with growth factors, cell migrating/scattering factors and anti-integrin β1 antibody at 4-5 days into the culturing process. The culturing process is conducted for an extended period of time, so that any latent red blood cells are eliminated from the islet culture. The islets then continue to proliferate to produce an amount of islets which is sufficient for transplantation into a diabetic patient, including a human, to provide extended normoglycaemia and islet regeneration to fully treat diabetes mellitus without the growth of fibroblasts.

What is claimed is:  
     
         1 . A method for in vitro culturing and proliferating isolated Langerhans islets endocrine cells so as to be suitable for transplantation comprising: 
 providing viable Langerhans islets endocrine cells including cells capable of differentiating into insulin producing cells,    providing a first culturing medium comprising a basal medium supplemented with serum, at least one radical scavenger selected from the group consisting of nicotinamide, mannitol or superoxide dismutase, at least one growth factor selected from the group consisting of: insulin transferring selenite (ITS), epidermal growth factor (EGF), platelet derived growth factor (PDGF), thrombin, Linoleic Acid-BSA, hydrocortisone and progesterone; and at least one antinecrosis or antiapoptosis factor selected from the group consisting of: insulin-like growth factor-I (IGF 1) and -II (IGF 2), vascular endothelial growth factor (VeGF);    culturing the Langerhans islets endocrine cells including cells capable of differentiating into insulin producing cells for a period of about one day in the first culturing medium to form a first culture growth,    collecting the first culture growth and incubating at room temperature in fresh Dulbecco modified Eagle medium (DMEM) or serum free basal medium with anti-integrin β1 antibody for 45˜120 minutes to form a second culture growth;    suspending the second culture growth in a matrix material to provide a 3 dimensional culture growth environment and adding a second culturing medium comprising the supplemental basal medium and further including, at least, another growth factor and then culturing for 1 or 2 days to provide a third culture growth dispersed in the matrix material;    providing a third culturing medium for culturing the third culture growth in the matrix material, the third culturing medium comprising the supplemented basal medium without VeGF, and optionally adding to the third culturing medium nerve growth factor (NGF) and hepatocyte growth factor (HGF) if the islets of the third culture growth appeared thick and the center of the islets appeared dark, or optionally adding to the third culturing medium NGF and anti-integrin β1 antibody if the islets appeared too spread out and culturing for a period of about one or two days to form a fourth culture growth;    collecting the islets from the matrix material and adding an enzyme to the collected islets and to any adhering gel and incubating for about 10 minutes to provide an incubated product; and    aspirating the incubated product back and forth numerous times causing the gel acted on by the enzyme to be removed from the islets thereby exposing the fibroblasts to the force created during the back and forth aspiration causing the fibroblasts to become separated from the surface of the islets to prepare fibroblast free islets.    
     
     
         2 . The method of  claim 1 , further providing a fourth culturing medium comprising the basal medium supplemented with serum, insulin-transferrin-sodium selenite (ITS), Linoleic Acid-BSA, thrombin, EGF, nicotinamide, VeGF, IGF-1, IGF-2, superoxide dismutase and mannitol; 
 culturing the fibroblast free islets for about an 8-12 hours to provide a fifth culture growth;    providing a fifth culturing medium comprising DMEM with anti-integrin β1 antibody and culturing the fifth culture growth for 45˜120 minutes at room temperature to form a sixth culture growth;    suspending the sixth culture growth in a matrix material to provide a 3 dimensional culture growth environment and adding a sixth culturing medium comprising the supplemented basal medium and further including, at least, another growth factor and then culturing for 1 or 2 days to provide a seventh culture growth dispersed in the matrix material;    providing a seventh culturing medium for culturing the seventh culture growth dispersed in the matrix material, the seventh culturing medium comprising the supplemented basal medium without VeGF, and optionally adding to the third culturing medium NGF and HGF if the islets of the third culture growth appeared thick and the center of the islets appeared dark, or optionally adding to the third culturing medium NGF and anti-integrin β1 antibody if the islets appeared too spread out and culturing for a period of about one or two days to form an eighth culture growth; and    collecting the islets from the matrix material and adding an enzyme to the collected islets and to any adhering gel and incubating for about 10 minutes to provide an incubated product; and    aspirating the incubated product back and forth numerous times causing the gel acted on by the enzyme to be removed from the islets thereby exposing the fibroblasts to the force created during the back and forth aspiration causing the fibroblasts to become separated from the surface of the islets to prepare an increased number of fibroblast free islets.    
     
     
         3 . The method of  claim 1  wherein the serum is obtained from the same species as that of the Langerhans islets  
     
     
         4 . The method of  claim 1  wherein the Langerhans islets are either from a rat and the serum used is about 10% rat serum or are from a human and the serum used is about 10% human serum.  
     
     
         5 . The method of  claim 1  wherein the growth factor added to the second culturing medium is pituitary extract.  
     
     
         6 . The method of  claim 2  wherein the growth factor added to the sixth culturing medium is pituitary extract.  
     
     
         7 . The method of  claim 1  wherein the viable Langerhans islets endocrine cells including cells capable of differentiating into insulin producing cells for proliferation are derived from a patient for autotransplantation.  
     
     
         8 . A method for removing fibroblasts growing from the surface of in vitro proliferated Langerhans islets, comprising: 
 providing a plurality of proliferated islets having fibroblasts growing therewith and collected from a gel proliferation matrix;    adding an enzyme to the collected islets and to any gel adhering to the surface of the collected islets and incubating to provide an incubated product; and    aspirating the incubated product back and forth numerous times causing the gel acted on by the enzyme to be removed from the islets thereby exposing the fibroblasts to the force being created during the back and forth aspiration causing the fibroblasts to become separated from the surface of the islets to prepare fibroblast free islets    
     
     
         9 . The method of  claim 8  wherein the enzyme is dispase  
     
     
         10 . A culture product of proliferated fibroblast free islets produced by the method of  claim 1 .  
     
     
         11 . Use of the proliferated fibroblast free islets produced by the method of  claim 1  in treating diabetes mellitis by transplanting the proliferated Langerhans islets endocrine cells into a patient suffering from diabetes mellitis.  
     
     
         12 . Use of the proliferated fibroblast free islets produced by the method of  claim 7  to treat diabetes mellitis and to regenerate islets by autotransplanting the proliferated Langerhans islets endocrine cells into a patient suffering from diabetes mellitis.