Use of collagenase in the preparation of neural stem cell cultures

Abstract

The invention provides a method for using collagenase to dissociate neural stem cells in neural stem cell cultures. The collagenase treatment results in an increased cell viability and an increased number of proliferated neural stem cells over time.

We claim:  
     
         1 . A method for the in vitro proliferation of a neural stem cell culture comprising the steps of: 
 (a) obtaining dissociated neural tissue in cell suspension, the suspension containing one or more multipotent neural stem cells capable of producing progeny that are capable of differentiating into neurons and glia;    (b) culturing the cell suspension in a culture medium containing at least one proliferation-inducing growth factor to proliferate the neural stem cells in (a) to generate a neural stem cell culture; and    (c) passaging the cell culture in (b) by treating the culture with an amount of a collagenase preparation effective to dissociate the cells in the culture and passing the cell culture to a culture medium containing at least one proliferation-inducing growth factor to further proliferate the neural stem cell culture.    
     
     
         2 . The method of    claim 1    wherein the amount of the collagenase preparation is between 18-180 mU/ml.  
     
     
         3 . The method of    claim 1    wherein the amount of the collagenase preparation is between 54-126 mU/ml.  
     
     
         4 . The method of    claim 1    wherein the collagenase preparation further comprises at least one molecule selected from the group consisting of sulfhydryl protease, clostripain, aminopeptidase, or combinations thereof.  
     
     
         5 . The method of    claim 1    wherein the collagenase preparation is substantially pure, and contains minimal secondary proteolytic activity.  
     
     
         6 . The process of    claim 1   , wherein the proliferation-inducing growth factor is selected from the group consisting of epidermal growth factor, amphiregulin, acidic fibroblast growth factor, basic fibroblast growth factor, transforming growth factor alpha, leukocyte inhibitory factor (LIF), glycostatin C and combinations thereof.  
     
     
         7 . The method of    claim 1    wherein the neural stem cell culture comprises genetically modified neural stem cells.  
     
     
         8 . The method of    claim 1   , further comprising the step of differentiating the neural stem cell culture of (c) to produce a cell culture comprising differentiated neural cells selected from the group consisting of astrocytes, neurons, oligodendrocytes, and combinations thereof.  
     
     
         9 . The method of    claim 1    or    claim 8   , further comprising contacting the neural stem cell culture with a biological agent, and determining the effects of the biological agent on cells in the culture.  
     
     
         10 . The method of    claim 1    wherein the neural stem cell culture is a suspension culture.  
     
     
         11 . The method of    claim 1    wherein the neural stem cell culture is an adhesion culture.  
     
     
         12 . The method of    claim 1    wherein the neural stem cell culture comprises human neural stem cells.  
     
     
         13 . A method for the in vitro proliferation of a neural stem cell culture comprising the steps of: 
 (a) obtaining dissociated neural tissue in cell suspension, the suspension containing one or more multipotent neural stem cells capable of producing progeny that are capable of differentiating into neurons and glia;    (b) culturing the cell suspension in a culture medium containing at least one proliferation-inducing growth factor to proliferate the neural stem cells in (a) to generate a neural stem cell culture; and    (c) passaging the cell culture in (b) by treating the culture with an amount of a collagenase preparation effective to dissociate the cells in the culture and passing the cell culture to a culture medium containing at least one proliferation-inducing growth factor to further proliferate the neural stem cell culture, wherein the percent viability of the cells in the culture is at least 60% after being passaged.    
     
     
         14 . The method of    claim 13   , wherein the percent viability of the cells in the culture is at least 75% after being passaged.  
     
     
         15 . The method of    claim 13   , wherein the percent viability of the cells in the culture is at least 85% after being passaged.  
     
     
         16 . The method of    claim 13   , wherein the amount of the collagenase preparation is between 18-180 mU/ml.  
     
     
         17 . The method of    claim 13   , wherein the amount of the collagenase preparation is between 54-126 mU/ml.  
     
     
         18 . The method of    claim 13   , wherein the collagenase preparation further comprises at least one molecule selected from the group consisting of sulfhydryl protease, clostripain, aminopeptidase, or combinations thereof.  
     
     
         19 . The method of    claim 13   , wherein the collagenase preparation is substantially pure, and contains minimal secondary proteolytic activity.  
     
     
         20 . The method of    claim 13   , wherein the neural stem cell culture comprises genetically modified neural stem cells.  
     
     
         21 . The method of    claim 13   , further comprising the step of differentiating the neural stem cell culture of (c) to produce a cell culture comprising differentiated neural cells selected from the group consisting of astrocytes, neurons, oligodendrocytes, and combinations thereof.  
     
     
         22 . The method of    claim 13    wherein the neural stem cell culture is a suspension culture.  
     
     
         23 . The method of    claim 13    wherein the neural stem cell culture is an adhesion culture.  
     
     
         24 . The method of    claim 13    wherein the neural stem cell culture comprises human neural stem cells.  
     
     
         25 . A cell culture made according to any one of claims  13 - 24 .  
     
     
         26 . A method for expanding a neural stem cell culture comprising the steps of: 
 (a) culturing a neural stem cell culture that contains one or more multipotent neural stem cells capable of producing progeny that are capable of differentiating into neurons and glia in a culture medium containing at least one proliferation-inducing growth factor to proliferate the culture; and    (b) passaging the cell culture in (a) by treating the culture with an amount of a collagenase preparation effective to dissociate the cells in the culture and passing the cell culture to a culture medium containing at least one proliferation-inducing growth factor to further proliferate the neural stem cell culture, wherein there is at least a three-fold increase in cell number between weekly passages.    
     
     
         27 . The method of    claim 26    wherein there is at least a five-fold increase in cell number between weekly passages.  
     
     
         28 . The method of    claim 26    wherein the neural stem cell culture is a suspension culture.  
     
     
         29 . The method of    claim 26    wherein the neural stem cell culture is an adhesion culture.  
     
     
         30 . The method of    claim 26    wherein the neural stem cell culture comprises human neural stem cells.  
     
     
         31 . A cell culture made according to any one of claims  26 - 30 .  
     
     
         32 . A method for expanding a neural stem cell culture comprising the steps of: 
 (a) culturing a neural stem cell culture that contains one or more multipotent neural stem cells capable of producing progeny that are capable of differentiating into neurons and glia in a culture medium containing at least one proliferation-inducing growth factor to proliferate the culture; and    (b) passaging the cell culture in (a) by treating the culture with an amount of a collagenase preparation effective to dissociate the cells in the culture and passing the cell culture to a culture medium containing at least one proliferation-inducing growth factor to further proliferate the neural stem cell culture, wherein the accumulated cell number can be expanded from 10 6  cells to at least 10 9  cells in less than 49 days.    
     
     
         33 . The method of    claim 32    wherein the accumulated cell number can be expanded from 10 6  cells to at least 10 9  cells in less than 42 days.  
     
     
         34 . The method of    claim 32    wherein the neural stem cell culture is a suspension culture.  
     
     
         35 . The method of    claim 32    wherein the neural stem cell culture is an adhesion culture.  
     
     
         36 . The method of    claim 32   , wherein the neural stem cell culture comprises human neural stem cells.  
     
     
         37 . A cell culture made according to any one of claims  32 - 36 .