Movement and Selection of Micro-Objects in a Microfluidic Apparatus

Abstract

A microfluidic apparatus is provided having one or more sequestration pens configured to isolate one or more target micro-objects by changing the orientation of the microfluidic apparatus with respect to a globally active force, such as gravity. Methods of selectively directing the movements of micro-objects in such a microfluidic apparatus using gravitational forces are also provided. The micro-objects can be biological micro-objects, such as cells, or inanimate micro-objects, such as beads.

1 . A microfluidic device comprising an enclosure, wherein said enclosure comprises:
 a flow path configured to contain a flow of a fluidic medium; and   a microfluidic sequestration pen comprising:
 an isolation region; and 
 a connection region fluidically connecting said isolation region to said flow path, wherein said connection region includes a tapered opening; 
   
       wherein said isolation region has a volume sufficient to hold at least one target micro-object, and wherein, when said microfluidic device is tilted such that said flow path is located below said sequestration pen, target micro-objects located in said isolation region are retained in said isolation region while target micro-objects located in said connection region settle into said flow path. 
     
     
         2 .- 5 . (canceled) 
     
     
         6 . The microfluidic device of  claim 1 , wherein said connection region includes an opening to said isolation region and wherein the opening of said connection region to said isolation region has a width of less than about 50 microns. 
     
     
         7 . The microfluidic device of  claim 1 , wherein said isolation region includes an opening that interfaces with said connection region, and wherein said sequestration pen further comprises an obstruction or a constriction located at the interface between said connection region and the opening of said isolation region. 
     
     
         8 .- 11 . (canceled) 
     
     
         12 . The microfluidic device of  claim 1 , wherein said isolation region has a length of at least about 25 microns. 
     
     
         13 . (canceled) 
     
     
         14 . The microfluidic device of n  claim 1 , wherein said microfluidic sequestration pen comprises a plurality of said isolation regions. 
     
     
         15 . The microfluidic device of  claim 14 , wherein each isolation region of said plurality extends laterally from said connection region. 
     
     
         16 . The microfluidic device of  claim 14 , wherein one isolation region of said plurality extends distally from a distal end of said connection region and the other isolation region(s) of said plurality extend laterally from a side of said connection region. 
     
     
         17 . (canceled) 
     
     
         18 . The microfluidic device of  claim 14 , wherein the opening of at least one isolation region of said plurality has a width or cross-sectional area that is smaller than other isolation regions of said plurality. 
     
     
         19 .- 21 . (canceled) 
     
     
         22 . The microfluidic device of  claim 1 , further comprising a plurality of said microfluidic sequestration pens, each comprising a corresponding isolation region and a corresponding connection region fluidically connecting said corresponding isolation region to said flow path. 
     
     
         23 . The microfluidic device of  claim 1 , further comprising a secondary isolation region fluidically connected with said isolation region, wherein, upon tilting said microfluidic device, a micro-object located within said isolation region can settle into said secondary isolation region. 
     
     
         24 .- 27 . (canceled) 
     
     
         28 . The microfluidic device of  claim 1 , wherein said flow path is defined by a channel, said channel further comprising a trap formed in a wall of said channel opposite an opening to said sequestration pen, said trap comprising an opening that is approximately equal to or greater than the diameter of said target micro-object. 
     
     
         29 .- 34 . (canceled) 
     
     
         35 . The microfluidic device of  claim 1 , further comprising a support structure and a microfluidic structure that together define said enclosure, wherein said support structure and said microfluidic structure are configured to support the generation of an electrokinetic force within at least a portion of said enclosure. 
     
     
         36 . The microfluidic device of  claim 35 , wherein the portion of said enclosure in which said electrokinetic force can be generated includes said sequestration pen. 
     
     
         37 .- 39 . (canceled) 
     
     
         40 . The device of  claim 1 , wherein a surface of said sequestration pen or sequestration pens of said plurality further comprises a functional moiety that specifically binds to said target micro-objects and wherein said functional moiety comprises a polymer, a carbohydrate, an antibody, an extracellular matrix component or derivative thereof, or any combination thereof. 
     
     
         41 .- 42 . (canceled) 
     
     
         43 . A method for loading a target micro-object into a microfluidic sequestration pen, the method comprising:
 flowing a fluidic medium containing target micro-objects into a flow path of a microfluidic device, wherein said device comprises a microfluidic sequestration pen fluidically connected to said flow path, wherein a surface of said microfluidic sequestration pen further comprises one or more functional moieties that specifically bind to said target micro-objects;   positioning said microfluidic device into a first loading position such that a first portion of fluidic medium in said flow path is located above an opening of said sequestration pen to said flow path;   allowing gravity to act on said target micro-objects for an amount of time sufficient for any target micro-objects located in the first portion of fluidic medium in said flow path to settle into said sequestration pen;   allowing said target micro-objects to bind said one or more functional moieties; and   moving any unbound target micro-objects from said microfluidic sequestration pen back into said flow path.   
     
     
         44 . The method of  claim 43 , further comprising slowing or substantially stopping the flow of said fluidic medium through said flow path prior to positioning said microfluidic device into the first loading position. 
     
     
         45 . (canceled) 
     
     
         46 . The method of  claim 43 , wherein positioning said microfluidic device into the first loading position comprises tilting said microfluidic device along an axis of said flow path. 
     
     
         47 .- 49 . (canceled) 
     
     
         50 . The method of  claim 43 , wherein said unbound target micro-objects are moved using dielectrophoresis (DEP). 
     
     
         51 . (canceled) 
     
     
         52 . The method of  claim 43 , wherein moving any unbound target micro-objects back into said flow path comprises:
 positioning said microfluidic device such that the first portion of fluidic medium in said flow path is located beneath the opening of said sequestration pen to said flow path; and   allowing gravity to act on any unbound target micro-objects remaining in said sequestration pen for an amount of time sufficient for such unbound target micro-objects to settle into the first portion of fluidic medium in said flow path.   
     
     
         53 .- 60 . (canceled) 
     
     
         61 . The method of  claim 43 , wherein said microfluidic device comprises a plurality of said sequestration pens, and wherein said plurality of sequestration pens are loaded with target micro-objects in parallel. 
     
     
         62 .- 68 . (canceled) 
     
     
         69 . A method for concentrating micro-objects in a microfluidic device, the method comprising:
 flowing a fluidic medium comprising micro-objects into a flow path of a microfluidic device, said microfluidic device comprising a concentration chamber opening off one side of said flow path, said concentration chamber having a chamfered opening and a distal end, wherein the distal end has a volume that is smaller than a volume of said chamfered opening;   arresting the flow of said fluidic medium within said flow path; and   allowing gravity to act on said micro-objects in said fluidic medium for an amount of time sufficient for said micro-objects to settle into and substantially fill the distal end of said concentration chamber.   
     
     
         70 . The method of  claim 69 , wherein said microfluidic device comprises a plurality of said concentration chambers. 
     
     
         71 .- 85 . (canceled) 
     
     
         86 . The method of  claim 43 , wherein said microfluidic sequestration pen is part of a microfluidic circuit having a cover, wherein said one or more functional moieties comprises two or more antibodies that specifically bind to said target micro-objects, wherein said two or more antibodies are disposed on an inside surface of the cover within the sequestration pen. 
     
     
         87 . The method of  claim 86 , wherein the target micro-objects are biological cells, and wherein allowing gravity to act on said target micro-objects provides a single biological cell loaded into the microfluidic sequestration pen, wherein the method further comprises:
 incubating the single biological cell for an amount of time sufficient to form a clonal population of cells;   wherein allowing said target micro-objects to bind said one or more functional moieties comprises allowing cells of said clonal population of cells to bind said two or more antibodies;   wherein moving any unbound target micro-objects from said microfluidic sequestration pen back into said flow path comprises:
 positioning said microfluidic device such that the first portion of fluidic medium in said flow path is located beneath the opening of said sequestration pen to said flow path; and 
 allowing gravity to act on any unbound cells of said clonal population of cells in said sequestration pen for an amount of time sufficient for such unbound cells of said clonal population of cells to settle into the first portion of fluidic medium in said flow path.