Manipulation of Cell Nuclei in a Micro-Fluidic Device

Abstract

Aspects of the present disclosure are directed to the manipulation of a cell nucleus in a micro-fluidic device as well as compositions, systems, and kits for performing such methods. In some aspects, the disclosure provides methods for placing one or more selected cell nuclei into an isolation region of a sequestration pen in a micro-fluidic device. The isolated nucleus/nuclei may then be retrieved from the isolation region of the sequestration pen and used in any desired downstream assay or process.

1 .- 58 . (canceled) 
     
     
         59 . A method of selecting a cell nucleus, said method comprising:
 identifying a cell nucleus associated with at least one characteristic, wherein the cell nucleus is identified within a microfluidic device comprising:
 a flow region containing a first liquid medium; 
 a first sequestration pen comprising an isolation region and a connection region, wherein the connection region has a proximal opening to the flow region and a distal opening to the isolation region and wherein the isolation region opens to the connection region; and 
 a substrate comprising a covalently linked coating material; 
 wherein the isolation region of the first sequestration pen is an unswept region of the flow region and the substrate is configured to selectively generate forces capable of moving cell nuclei; and 
   repositioning the cell nucleus within the microfluidic device using dielectrophoretic force responsive to identifying that the cell nucleus is associated with the at least one characteristic.   
     
     
         60 . The method of  claim 59 , wherein the microfluidic device comprises a plurality of inner surfaces, wherein the plurality of inner surfaces has been treated with a blocking solution to prevent or reduce nuclei adherence. 
     
     
         61 . The method of  claim 60 , wherein the blocking solution comprises a polymer comprising alkylene ether moieties. 
     
     
         62 . The method of  claim 61 , wherein the polymer comprises polyethylene glycol. 
     
     
         63 . The method of  claim 60 , wherein the blocking solution comprises a polymer comprising saccharide moieties. 
     
     
         64 . The method of  claim 63 , wherein the blocking solution comprises dextran. 
     
     
         65 . The method of  claim 60 , wherein the blocking solution comprises a polymer comprising amino acid moieties. 
     
     
         66 . The method of  claim 65 , wherein the blocking solution comprises albumin. 
     
     
         67 . (canceled) 
     
     
         68 . The method of  claim 59 , wherein the coating material comprises molecules having a linking group and an alkyl moiety, wherein the linking group is covalently bonded to the substrate surface. 
     
     
         69 . The method of  claim 68 , wherein the alkyl moiety is a fluoroalkyl group. 
     
     
         70 . The method of  claim 69 , wherein the alkyl moiety is a perfluoroalkyl group. 
     
     
         71 . (canceled) 
     
     
         72 . The method of  claim 68 , wherein the alkyl moiety comprises a linear chain of carbons comprising at least 10 carbon atoms. 
     
     
         73 . The method of  claim 68 , wherein the molecules of the coating material form a densely-packed monolayer structure. 
     
     
         74 . The method of  claim 68 , wherein the coating material comprises molecules having a linking group and a cationic moiety and/or an anionic moiety, wherein the linking group is covalently bonded to the substrate surface. 
     
     
         75 . The method of  claim 74 , wherein the cationic moiety comprises a quaternary ammonium group. 
     
     
         76 . The method of  claim 74 , wherein the anionic moiety comprises a phosphonic acid, carboxylic acid, or sulfonic acid. 
     
     
         77 . The method of  claim 74 , wherein the coating material comprises molecules having a linking group and a zwitterionic moiety. 
     
     
         78 . The method of  claim 77 , wherein the zwitterionic moiety is selected from carboxybetaines, sulfobetaines, sulfamic acids, and amino acids. 
     
     
         79 . The method of  claim 68 , wherein the coating material comprises a polymer comprising alkylene ether moieties, saccharide moieties, or amino acid moieties. 
     
     
         80 . The method of  claim 79 , wherein the coating material comprises dextran. 
     
     
         81 . The method of  claim 79 , wherein the coating material comprises poly-ethylene glycol. 
     
     
         82 . The method of  claim 79 , wherein the coating material comprises albumin. 
     
     
         83 . The method of  claim 59 , wherein the at least one characteristic comprises a morphology of the cell nuclei. 
     
     
         84 . The method of  claim 59 , wherein the at least one characteristic includes detectable labelling of the cell nucleus by a binding agent. 
     
     
         85 . The method of  claim 84 , wherein the at least one characteristic comprises an intensity of the detectable labelling by the binding agent. 
     
     
         86 . The method of  claim 59 , wherein the at least one characteristic are identified using a machine learning algorithm. 
     
     
         87 . The method of  claim 59 , wherein repositioning the cell nucleus in the microfluidic device comprises moving the cell nucleus from the flow region to the first sequestration pen using dielectrophoretic force. 
     
     
         88 . The method of  claim 59 , wherein the microfluidic device comprises a second sequestration pen, and repositioning the cell nucleus in the microfluidic device comprises moving the cell nucleus from the first sequestration pen to the second sequestration pen using dielectrophoretic force. 
     
     
         89 . The method of  claim 59 , wherein repositioning the cell nuclei in the microfluidic device comprises moving the cell nuclei to a portion of the microfluidic device for export using dielectrophoretic force. 
     
     
         90 . The method of  claim 59 , wherein repositioning the cell nuclei in the microfluidic device comprises moving the cell nuclei to a portion of the microfluidic device configured for electrowetting using dielectrophoretic force. 
     
     
         91 . The method of  claim 59 , wherein the at least one characteristic is a combination of more than one characteristic.