Methods for multiplex analytical measurements in single cells of solid tissues

Abstract

The invention provides a method for the isolation of a single cell embedded in a tissue while preserving the state of molecules of the cell, and therefore allows for transformation of a single target cell in live tissue into a format that can be evaluated using analytical methods.

1 . A method of lysing a single cell present in a tissue, the method comprising:
 a) identifying a cell from a tissue;   b) contacting a detergent-containing lysis buffer with the intracellular space of the identified cell;   c) allowing the lysis buffer to spread within the intracellular space of the identified cell for a period of time, wherein the cell is lysed from the inside of the cell; and   d) collecting the lysate.   
     
     
         2 - 12 . (canceled) 
     
     
         13 . The method of  claim 1 , wherein the method occurs in the absence of tissue fixation and tissue disaggregation. 
     
     
         14 . The method of  claim 1 , wherein the isolated cell is in an organotypic culture. 
     
     
         15 . The method of  claim 1 , wherein the lysate is collected by suctioning the lysate using a suction channel. 
     
     
         16 . The method of  claim 15 , wherein the suction channel is a bent suction micropipette. 
     
     
         17 . The method of  claim 1 , wherein the collected lysate is further applied to a nitrocellulose pad. 
     
     
         18 . The method of  claim 17 , wherein a standard is also applied to the nitrocellulose pad. 
     
     
         19 . The method of  claim 1 , wherein the lysate is evaluated using an analytical method. 
     
     
         20 . The method of  claim 19 , wherein the analytical method is selected from the group consisting of mass spectrometry, protein microarray, RT-qPCR, RNA-Seq, and MALDI-MS. 
     
     
         21 . The method of  claim 1 , wherein the cell is part of a live solid tissue. 
     
     
         22 . The method of  claim 1 , wherein the detergent is sodium dodecyl sulfate. 
     
     
         23 . A method of analyzing a cell present in a tissue, the method comprising:
 a) identifying a cell from a tissue;   b) contacting a detergent-containing lysis buffer to the intracellular space of the identified cell;   c) allowing the lysis buffer to spread within the intracellular space of the identified cell for a period of time, wherein the cell is lysed from the inside of the cell;   d) collecting the lysate;   e) applying the collected lysate to a solid support; and   f) evaluating the collected lysate using an analytical method.   
     
     
         24 . The method of  claim 1 , wherein the outer cell membrane of the identified cell is intact or mostly intact prior to lysate collection. 
     
     
         25 . The method of  claim 23 , wherein the outer cell membrane of the identified cell is intact or mostly intact prior to lysate collection. 
     
     
         26 . A composition comprising a tissue, a cell present in the tissue having an intracellular space, and a lysis buffer present in the tissue, wherein all or most of the lysis buffer is confined within the intracellular space. 
     
     
         27 . The composition of  claim 26 , wherein the lysis buffer comprises a detergent. 
     
     
         28 . The composition of  claim 27 , wherein the detergent is sodium dodecyl sulfate. 
     
     
         29 . A composition comprising a tissue, at least two cells present in the tissue, and a lysis buffer present in the tissue, wherein each of the at least two cells has an intracellular space, and wherein all or most of the lysis buffer is confined within the intracellular spaces of the at least two cells. 
     
     
         30 . The composition of  claim 29 , wherein the lysis buffer comprises a detergent. 
     
     
         31 . The composition of  claim 30 , wherein the detergent is sodium dodecyl sulfate. 
     
     
         32 . A robotic system comprising (a) a first robotic arm comprising a first microcapillary configured to contact a lysis buffer with an intracellular space of a cell through a first aperture, thereby producing a lysate, (b) a second robotic arm comprising a second microcapillary configured to collect the lysate through a second aperture, (c) and a computer coupled to the first and second robotic arms and programmed to control the first and second robotic arms. 
     
     
         33 . The robotic system of  claim 32 , wherein the second aperture is larger than the first aperture. 
     
     
         34 . The robotic system of  claim 32 , wherein the first aperture is about 0.01 μm to 1 μm. 
     
     
         35 . The robotic system of  claim 32 , wherein the second aperture is about 1 μm to 10 μm. 
     
     
         36 . A system comprising at least two robotic systems of  claim 32 , wherein each of the at least two robotic systems is centrally controlled from a single user interface.