Method for processing tissue samples

Abstract

This disclosure provides methods for producing a sample of subcellular organelles, particularly nuclei, from a tissue. In some embodiments, this disclosure provides a method of processing a tissue sample involves performing enzymatic/chemical disruption of tissue in a chamber to produce disrupted tissue comprising released cells and/or nuclei and debris; separating the released cells and/or nuclei from the debris therein; and moving the released cells and/or nuclei. In some instances, the method comprises mechanical disruption of the tissue sample.

1 . A system comprising:
 (a) an instrument comprising:
 (i) one or more cartridge interfaces configured to engage a cartridge; 
 (ii) a fluidics module comprising:
 (1) one or more containers containing one or more liquids and/or gasses; 
 (2) one or more fluid lines connecting the containers with fluid ports in the cartridge interface; and 
 (3) one or more pumps configured to move liquids and/or gasses into and/or out of the fluid port(s); 
 
 (iii) a mechanical module comprising an actuator; 
 (iv) optionally, a magnetic processing module comprising a source of magnetic force, wherein the magnetic force is positioned to form a magnetic field in the processing chamber; 
 (v) optionally, a measurement module; 
 (vi) optionally, a control module comprising a processor and memory, wherein the memory comprises code that, when executed by the processor, operates the system; and 
   (b) one or more cartridges, each engaged with one of the cartridge interfaces, wherein each cartridge comprises:
 (i) a sample inlet port; 
 (ii) one or more cartridge ports communicating with the fluid ports in the cartridge interface; 
 (iii) a preprocessing chamber communicating with the sample inlet port and with at least one cartridge port, and comprising a tissue disruptor configured for mechanical disruption of tissue, wherein the tissue disruptor engages with and is actuated by the actuator when the cartridge is engaged with the cartridge interface; 
 (iv) a strain chamber communicating with the preprocessing chamber configured to separate cells and/or nuclei from disrupted tissue; 
 (v) a processing chamber communicating with the strain chamber, optionally communicating with one or more cartridge ports and optionally configured to perform one or more processing steps on separated cells and/or nuclei; and 
 (vi) optionally, one or more waste chambers fluidically connected with the processing chamber. 
   
     
     
         2 . The system of  claim 1 , wherein the tissue disruptor comprises a grinder, a pestle, or a variable orifice. 
     
     
         3 . The system of  claim 1 , further comprising a barcode reader. 
     
     
         4 . The system of  claim 1 , comprising a measurement module (v) that performs optical imaging to measure titer, clumping, and/or viability of cells or nuclei. 
     
     
         5 . The system of  claim 1 , comprising a measurement module (v) and a control system (vi), wherein the measurement module measures, and one or more time points, characteristics of a sample in the processing chamber, and control system comprises code that determines a state of the sample, e.g., viability or degree of single cell or nuclei dissociation, and that adjusts processing parameters. 
     
     
         6 . The system of  claim 1 , further comprising:
 (c) an analysis module, wherein an input port of the analysis module is in fluid communication with the processing chamber.   
     
     
         7 . The system of  claim 6 , wherein the analysis module performs an analysis selected from one or more of: DNA sequencing, next generation DNA sequencing, next next generation DNA sequencing, proteomic analysis, genomic analysis, gene expression analysis, gene mapping, carbohydrate characterization and profiling, lipid characterization and profiling, flow cytometry, imaging, DNA or RNA microarray analysis, metabolic profiling, functional analysis, and mass spectrometry. 
     
     
         8 . The system of  claim 1 , wherein the cartridge interface comprises a means of positioning the cartridge in the instrument that engages the fluidic module and the mechanical module and optionally is temperature controlled. 
     
     
         9 . (canceled) 
     
     
         10 . A method comprising:
 (a) providing a tissue sample to a preprocessing chamber;   (b) automatically performing mechanical and enzymatic/chemical disruption of the tissue in the preprocessing chamber to produce disrupted tissue comprising released cells and/or nuclei and debris;   (c) automatically moving the disrupted tissue into a strain chamber comprising a strainer and/or filter and separating the released cells and/or nuclei from the debris therein; and   (d) automatically moving the released cells and/or nuclei into a processing chamber.   
     
     
         11 . The method of  claim 9 , wherein (d) further comprises performing at least one processing step on the released cells and/or nuclei in the processing chamber. 
     
     
         12 . The method of  claim 11 , wherein processing comprises one or more automatically performed processes selected from:
 (I) lysing cells;   (II) capturing cells;   (III) isolating nucleic acid;   (IV) isolating protein;   (V) converting RNA into cDNA;   (VI) preparing one or more libraries of adapter tagged nucleic acids;   (VII) performing PCR;   (VIII) isolating individual cells or individual nuclei in nanodrops or nanoboluses; and   (IX) outputting released cells and/or nuclei into output vessels such as 8 well strip tubes, microtiter plates, tubes, a chamber in the cartridge, or other vessels capable of receiving cell suspensions.   
     
     
         13 . The method of  claim 9 , further comprising:
 (e) automatically capturing the released cells and/or nuclei in the processing chamber by binding to magnetically attractable particles comprising moieties having affinity for the cells and/or nuclei and applying a magnetic force to the processing chamber to immobilize the captured cells and/or nuclei; and   (f) automatically capturing debris in the processing chamber by binding to magnetically attractable particles comprising moieties having affinity for cell and/or nuclei debris and applying a magnetic force to the processing chamber to immobilize the captured cells and/or nuclei debris.   
     
     
         14 . The method of  claim 9 , further comprising:
 (e) automatically monitoring cell and/or nuclei titer in the processing chamber and, when the titer reaches a desired level, exchanging a dissociation solution used to dissociate the tissue for a buffer.   
     
     
         15 . A cartridge comprising:
 (i) a sample inlet port;   (ii) one or more cartridge ports configured to communicate with fluid ports in a cartridge interface;   (iii) a preprocessing chamber communicating with the sample inlet port and with at least one cartridge port, and comprising a tissue disruptor configured for mechanical disruption of tissue, wherein the tissue disruptor engages with and is actuated by the actuator when the cartridge is engaged with the cartridge interface;   (iv) a strain chamber communicating with the preprocessing chamber configured to separate cells from disrupted tissue;   (v) a processing chamber communicating with the strain chamber, optionally communicating with one or more cartridge ports and configured to perform one or more processing steps on separated cells; and   (vi) optionally, one or more waste chambers fluidically connected with the processing chamber.   
     
     
         16 . The cartridge of  claim 15 , further comprising a cap that opens and closes the sample inlet port. 
     
     
         17 . The cartridge of  claim 16 , wherein the cap comprises a tissue disruptor element that moves about rotationally and back and forth along an axis. 
     
     
         18 . (canceled) 
     
     
         19 . (canceled) 
     
     
         20 . The cartridge of  claim 18 , wherein the holder comprises a mesh screen. 
     
     
         21 . The cartridge of  claim 18 , further comprising a grinding element for grinding tissue in the preprocessing chamber. 
     
     
         22 . (canceled) 
     
     
         23 . The cartridge of  claim 15 , further comprising a plunger configured to move slideably within the preprocessing chamber. 
     
     
         24 . (canceled) 
     
     
         25 . (canceled) 
     
     
         26 . (canceled) 
     
     
         27 . (canceled)