Neuron cultivation device, neuron cultivating method, cultivated neuron, analysis and identification of protein in axon bundle, and usage of cultivated neuron

Abstract

An object is to provide a neuron cultivation device which promptly develops bundles of axons extending from neurons in vitro. A device for cultivating neuron with axon, the device comprising a cultivation plate and a plurality of modules arranged in the cultivation plate. Each of the modules includes at least one of first chambers receivable of cell bodies of neurons, at least one of second chambers, and at least one of channels receivable of a bundle of axon extended from the cell bodies. The channels connect the first chambers and the second chambers. Bottom ends of the first chambers, the second chambers and the channels are closed and top ends of the first chambers and the second chambers are open.

We claim: 
     
         1 . A device for cultivating an aggregated neuron body consisting of a plurality of neurons with cell bodies and axons, the aggregated neuron body including a spheroid and a bundle of axons extended from the spheroid in one direction, the device comprising:
 a cultivation plate ( 10 ); and   a plurality of modules ( 11 ) arranged in the cultivation plate ( 10 ), each of the modules ( 11 ) including a first chamber ( 12   a ) receivable of cell bodies of neurons, a second chamber ( 12   b ), and a channel ( 13 ) receivable of the bundle of axon extended from the cell bodies, the channel ( 13 ) connecting the first chamber ( 12   a ) and the second chamber ( 12   b ),   wherein bottom ends of the first chamber ( 12   a ), the second chamber ( 12   b ) and the channel ( 13 ) are closed, top ends of the first chamber ( 12   a ) and the second chamber ( 12   b ) are open, and the channel ( 13 ) is 100-150 μm in width, 100-200 μm in height and 1 mm or more in length.   
     
     
         2 . The device according to  claim 1 , wherein culture fluid ( 18 ) is received in the first chamber ( 12   a ), the second chamber ( 12   b ) and the channel ( 13 ), the top end of the second chamber ( 12   b ) is closed by at least one of seal members ( 17 ). 
     
     
         3 . The device according to  claim 1 , wherein the second chamber ( 12   b ) is receivable of skeletal muscle, so that the bundle of axon extended from the cell bodies conjugates with the skeletal muscle. 
     
     
         4 . A method for cultivating an aggregated neuron body consisting of a plurality of neurons with cell bodies and axons, the aggregated neuron body including a spheroid and a bundle of axons extended from the spheroid in one direction, the method comprising steps of:
 applying cultivation fluid in a first chamber ( 12   a ), a second chamber ( 12   b ), and a channel ( 13 ) of 100-150 μm in width, 100-200 μm in height and 1 mm or more in length and connecting the first chamber ( 12   a ) and the second chamber ( 12   b ), the first chamber ( 12   a ), the second chamber ( 12   b ) and the channel ( 13 ) included in at least one of modules ( 11 ) arranged in a cultivation plate ( 10 );   inoculating one spheroid in the first chamber ( 12   a ); and   cultivating the neurons, so that the bundle of axons grows and extends from the spheroid in the channel ( 13 ).   
     
     
         5 . The method according to  claim 4 , wherein skeletal muscle is inoculated in the second chambers ( 12   b ), so that the bundle of axon extended from the spheroid conjugates with the skeletal muscle. 
     
     
         6 . A method of confirming purity in a bundle of axons of an aggregated neuron body consisting of a plurality of neurons with cell bodies and axons, the aggregated neuron body including a spheroid and the bundle of axons extended from the spheroid in one direction, the method comprising steps of:
 cultivating neurons so that one bundle of axons grows and extends from the spheroid, so that the aggregated neuron body includes the spheroid and the bundle of axons of 1 mm or more in length extended from the spheroid in the direction; and   confirming that the bundle of axons, said cell bodies being non-existent in the bundle of axons to a degree that markers indicating existence of the cell bodies in Western blotting are negative, through analyzing and identifying proteins in the bundle of axons.   
     
     
         7 . The method according to  claim 6 , wherein the markers are Map 2 and Nucleoporin. 
     
     
         8 . A method of analyzing and identifying proteins in a bundle of axons of an aggregated neuron body consisting of a plurality of neurons with cell bodies and axons, the aggregated neuron body including a spheroid and the bundle of axons extended from the spheroid in one direction, the method comprising steps of:
 cultivating the neurons so that one bundle of axons grows and extends from the spheroid;   slicing the bundle of axons; and   analyzing and identifying protein existing in the bundle of axons.   
     
     
         9 . A method of using an aggregated neuron body consisting of a plurality of neurons with cell bodies and axons, the aggregated neuron body including a spheroid and a bundle of axons extended from the spheroid in one direction, the method comprising steps of:
 cultivating the neurons so that the bundle of axons grows and extends from the spheroid;   applying fluorescent treatment to the neurons; and   stimulating the neurons.   
     
     
         10 . The method according to  claim 9 , wherein applying fluorescent treatment to the neurons is making the neurons incorporate calcium-responsive fluorescent dye. 
     
     
         11 . The method according to  claim 9 , wherein stimulating the neurons is providing KCl to the cell bodies so as to observe electrophysiologically activity. 
     
     
         12 . A method of analyzing and identifying proteins in a bundle of axons of an aggregated neuron body consisting of a plurality of neurons with cell bodies and axons, the aggregated neuron body including a spheroid and the bundle of axons extended from the spheroid in one direction, the method comprising steps of:
 cultivating the neurons so that one bundle of axons grows and extends from the spheroid;   slicing the bundle of axons; and   analyzing and identifying protein existing in the bundle of axons by immunostaining.