System for monitoring cell motility in real-time

Abstract

The invention relates to devices, devices for arraying biomolecules, including cells, methods for arraying biomolecules, assays for monitoring cellular movement, and systems for monitoring cellular movement. The devices include a support; a first layer configured to be placed in fluid-tight contact with the support, the first layer having an upper surface and defining a pattern of micro-orifices, each micro-orifice of the pattern of micro-orifices having walls and defining a micro-region on the support when the first layer is placed in fluid-tight contact with the support such that the walls of said each micro-orifice and the micro-region on the support together define a micro-well; and a second layer configured to be placed in fluid-tight contact with the upper surface of the first layer, the second layer defining a pattern of macro-orifices, each macro-orifice of the pattern of macro-orifices having walls and defining a macro-region when the first layer is placed in fluid-tight contact with the support and the second layer is placed in fluid-tight contact with the first layer such that the walls of the macro-orifice and the macro-region together define a macro-well.

1 . A method for monitoring cell motility comprising: 
 a) positioning a first layer to be in fluid-tight contact with a support, the first layer having an upper surface and defining a pattern of micro-orifices, each micro-orifice of the pattern of micro-orifices having walls and defining a micro-region on the support when the first layer is placed in fluid-tight contact with the support such that the walls of said each micro-orifice and the micro-region on the support together define a micro-well;    b) positioning a second layer to be in fluid-tight contact with an upper surface of the first layer, the second layer defining a pattern of macro-orifices, each macro-orifice of the pattern of macro-orifices having walls and defining a macro-region when the first layer is placed in fluid-tight contact with the support and the second layer is placed in fluid-tight contact with the first layer such that the walls of the macro-orifice and the macro-region together define a macro-well; each macro-region encompassing at least one micro-region;    c) immobilizing at least one cell of a plurality of cells in each respective micro-region on the support so as to situate the at least one cell within a corresponding micro-well, the cells thereby being arrayed on the support in a pattern that corresponds to the pattern of the micro-orifices;    d) allowing the cells to grow to confluency within the micro-regions;    e) providing at least one of a plurality of test agents to at least one macro-well and allowing said test agent to contact confluent cells;    f) removing said first and second layer;    g) monitoring cells for movement away from said micro-regions; said monitoring comprising imaging the cells for at least two different time points to generate an image for each of the at least two different time points to generate at least two images; and    h) calculating cellular movement from a comparison of the at least two images.    
     
     
         2 . A method for monitoring cell growth comprising: 
 a) positioning a first layer to be in fluid-tight contact with a support, the first layer having an upper surface and defining a pattern of micro-orifices, each micro-orifice of the pattern of micro-orifices having walls and defining a micro-region on the support when the first layer is placed in fluid-tight contact with the support such that the walls of said each micro-orifice and the micro-region on the support together define a micro-well;    b) positioning a second layer to be in fluid-tight contact with an upper surface of the first layer, the second layer defining a pattern of macro-orifices, each macro-orifice of the pattern of macro-orifices having walls and defining a macro-region when the first layer is placed in fluid-tight contact with the support and the second layer is placed in fluid-tight contact with the first layer such that the walls of the macro-orifice and the macro-region together define a macro-well; each macro-region encompassing at least one micro-region;    c) immobilizing at least one cell of a plurality of cells in each respective micro-region on the support so as to situate the at least one cell within a corresponding micro-well, the cells thereby being arrayed on the support in a pattern that corresponds to the pattern of the micro-orifices;    d) allowing the cells to grow to confluency within the micro-regions; providing at least one of a plurality of test agents to at least one macro-well and allowing said test agent to contact confluent cells;    e) removing said first and second layer;    f) monitoring cell growth, said monitoring comprising monitoring cell movement away from said micro-regions and further comprising imaging the cells for at least two different time points to generate an image for each of the at least two different time points to generate at least two images; and    g) calculating cellular growth from a comparison of the at least two images.    
     
     
         3 . A method for monitoring cell proliferation comprising: 
 a) positioning a first layer to be in fluid-tight contact with a support, the first layer having an upper surface and defining a pattern of micro-orifices, each micro-orifice of the pattern of micro-orifices having walls and defining a micro-region on the support when the first layer is placed in fluid-tight contact with the support such that the walls of said each micro-orifice and the micro-region on the support together define a micro-well;    b) positioning a second layer to be in fluid-tight contact with an upper surface of the first layer, the second layer defining a pattern of macro-orifices, each macro-orifice of the pattern of macro-orifices having walls and defining a macro-region when the first layer is placed in fluid-tight contact with the support and the second layer is placed in fluid-tight contact with the first layer such that the walls of the macro-orifice and the macro-region together define a macro-well; each macro-region encompassing at least one micro-region;    c) immobilizing at least one cell of a plurality of cells in each respective micro-region on the support so as to situate the at least one cell within a corresponding micro-well, the cells thereby being arrayed on the support in a pattern that corresponds to the pattern of the micro-orifices;    d) allowing the cells to grow to confluency within the micro-regions;    e) providing at least one of a plurality of test agents to at least one macro-well and allowing said test agent to contact confluent cells;    f) removing said first and second layer;    g) monitoring cells for multiplication, said monitoring comprising monitoring cell movement away from said micro-regions and further comprising imaging the cells for at least two different time points to generate an image for each of the at least two different time points to generate at least two images; and    h) calculating cellular multiplication from a comparison of the at least two images.    
     
     
         4 . An image processing method comprising, from captured image data: 
 a) creating a first histogram of image data signal strength along a first axis of the image data;    b) identifying first coarse island locations from the first histogram;    c) marking interstitial boundaries on the first axis between the first coarse island locations;    d) creating a second histogram of image data signal strength along a second axis of the image data;    e) identifying second coarse island locations from the second histogram; and    f) marking second interstitial boundaries on the second axis between the second coarse island locations.    
     
     
         5 . The image processing method of  claim 4 , wherein the first and second coarse island locations are determined from maxima of the first and second histograms respectively.  
     
     
         6 . The image processing method of  claim 4 , wherein the first and second coarse island locations are determined from portions of the first and second histograms respectively that exceed a predetermined threshold value.  
     
     
         7 . The image processing method of  claim 4 , wherein the first and second interstitial boundaries are marked at midpoints between the first and second coarse island locations respectively.  
     
     
         8 . The image processing method of  claim 4 , further comprising defining a plurality of island bounding boxes based on the first and second interstitial boundaries.  
     
     
         9 . An image processing method for biological testing purposes, comprising, from source image data representing imaged cellular material: for each pixel in a portion of the source image data, 
 determining whether the source image data indicates the presence of cellular material in a region of a scanning circle, and    if so, setting image data for a co-located, similarly dimensioned scanning circle in second image data; and    thereafter, identifying objects based on the second image data.    
     
     
         10 . The image processing method of  claim 9 , further comprising defining a bounding box for each object identified in the image data.