Nerve culture system

Abstract

Techniques and systems are disclosed for a bioassay that is an in vitro mimic of peripheral nerve generation using the sensory neurons that innervate the peripheral nervous system. In some embodiments, the techniques may assist in detecting the bioactivity or potency of nerve grafts (e.g., processed, acellular human allografts) for fostering or supporting peripheral nerve regeneration. In various embodiments, techniques comprise affixing neurons (e.g., a DRG) to a nerve graft segment to form a test construct; culturing the test construct in a medium; analyzing the test construct to indicate the amount of outgrowing nerve structure; and determining the potency of the nerve graft from a metric derived from the analysis. In some embodiments, techniques and materials may be used to test the effect of a varied test condition on nerve growth.

What is claimed is: 
     
         1 . A method for conducting a bioassay of a nerve graft, the method comprising:
 affixing a neuron or group of neurons to a first end of a nerve graft segment to form a test construct;   culturing the test construct in a medium for a period of time to allow nerve outgrowth from the neuron into the nerve graft segment;   performing an analysis of the test construct, wherein the analysis indicates an amount of an outgrowing nerve structure; and   determining a potency of the nerve graft from a metric derived from the analysis.   
     
     
         2 . The method of  claim 1 , wherein performing the analysis of the test construct comprises:
 preparing the test construct for sectioning;   sectioning the test construct into a plurality of sections, wherein each of the plurality of sections is removed longitudinally at a predetermined distance from a side of the nerve graft segment perpendicular to the first end;   staining each section with one or more stains to identify the outgrowing nerve structure from the neuron; and   determining a distance from the first end of the nerve graft segment to the end of a farthest-outgrowing nerve structure.   
     
     
         3 . The method of  claim 2 , wherein preparing the test construct for sectioning comprises fixing the test construct and paraffin-embedding the test construct. 
     
     
         4 . The method of  claim 2 , wherein one of the one or more stains is selected from the group consisting of βIII Tubulin, PGP9.5, and S100 antibody. 
     
     
         5 . The method of  claim 1 , wherein performing the analysis of the test construct comprises:
 scanning all or part of the test construct with diffusion tensor imaging and producing a tractography image wherein the outgrowing nerve structure is identified by the tractography image; and   quantifying the amount of outgrowing nerve structure from the tractography image.   
     
     
         6 . The method of  claim 1 , wherein the neurons are a dorsal root ganglia (DRG) explant. 
     
     
         7 . The method of  claim 1 , wherein performing the analysis of the test construct comprises assaying to determine the amount of a target protein associated with the outgrowing nerve structure. 
     
     
         8 . The method of  claim 7 , wherein the target protein is selected from the group consisting of βIII-Tubulin, S100, and GAP-43. 
     
     
         9 . The method of  claim 1 , wherein the outgrowing nerve structure is a neurite or a Schwann cell. 
     
     
         10 . The method of  claim 1 , further comprising passivating the nerve graft segment prior to affixing the neuron onto the nerve graft segment. 
     
     
         11 . The method of  claim 1 , wherein the period of time for culturing is from about 3 to about 7 days. 
     
     
         12 . The method of  claim 1 , wherein the affixing of the neuron onto the nerve graft segment comprises securing the neuron to the nerve graft segment with a collagen gel. 
     
     
         13 . The method of  claim 1 , wherein the nerve graft segment is taken from an acellular, processed nerve allograft. 
     
     
         14 . A method for testing the effect of a condition on nerve out growth, the method comprising:
 treating a nerve graft segment in accordance with a study protocol;   affixing a neuron to a first end of a nerve graft segment to form a test construct;   culturing the test construct in a medium for a period of time to allow nerve outgrowth from the neuron into the nerve graft segment;   performing an analysis of the test construct, wherein the analysis indicates an amount of an outgrowing nerve structure; and   determining the effect of the study protocol by comparing one or more analysis metrics derived from the analysis to one or more control metrics determined from nerve graft segments not treated with the study protocol.   
     
     
         15 . The method of  claim 14 , wherein the study protocol comprises one or more of:
 submerging the nerve graft segment in solution comprising a test compound;   dosing the nerve graft segment with radiation or heat;   dosing the nerve graft segment with an outgrowth inhibiting agent;   mechanically modifying the microstructure or macrostructure of the nerve graft segment;   dosing the nerve graft segment with an outgrowth promoting agent;   applying an electrical field to the nerve graft segment; and   seeding the nerve graft segment with stem cells or Schwann cells.   
     
     
         16 . The method of  claim 15 , wherein the outgrowth inhibiting agent is selected from the group consisting of:
 agents that degrade proteins;   agents that disrupt the microstructure or macrostructure of the nerve graft segment;   materials that modify the chemical structure of the nerve graft segment; and   functional blocking antibodies.   
     
     
         17 . The method of  claim 14 , wherein performing the analysis of the test construct comprises:
 scanning all or part of the test construct with diffusion tensor imaging and producing a tractography images wherein the outgrowing nerve structure is identified by the tractography images; and   quantifying the amount of outgrowing nerve structure from the tractography image.   
     
     
         18 . The method of  claim 14 , wherein the neuron is a DRG. 
     
     
         19 . The method of  claim 14 , wherein the outgrowing nerve structure is a neurite or a Schwann cell. 
     
     
         20 . The method of  claim 14 , wherein the nerve graft segment is taken from an acellular, processed nerve allograft. 
     
     
         21 . A kit comprising the nerve graft segment and the medium of  claim 14 .