Method for spatially barcoding cells in tissue slices

Patent metadata

Field	Value
Publication number	US-2022251632-A1
Application number	US-202017629255-A
Country	US
Kind code	A1
Filing date	Jul 22, 2020
Priority date	Jul 23, 2019
Publication date	Aug 11, 2022
Grant date	—

Abstract

Substrates and methods for spatially resolved analyses of tissue samples are provided, allowing the data from such analyses to be mapped back to the tissue's initial architecture. For analyses that have previously been conducted on bulk, homogenized samples or on dissociated cell components not directly traceable to their prior positions in the tissue sample, spatial resolution of this data can allow better characterization of cell-cell and cell-microenvironment relationships and interactions. This spatial context can aid in determining structure-function relationships in healthy and diseased tissues may thereby enhance the scientific understanding of tissue homeostasis, development, disease, and repair. The information determined using the methods of the disclosure can also be applied to enhancing diagnostics evaluation of diseased tissues.

First claim

1 . A substrate for spatially barcoding a tissue sample, comprising a matrix, wherein the matrix comprises:
(a) a plurality of discrete labeling regions arranged in a pattern, wherein the labeling regions are separated by non-labeling regions, and wherein each labeling region comprises one or more barcode labels, wherein the one or more barcode labels can be transferred from the matrix into a tissue sample upon contacting the substrate with the tissue sample; and (b) a support.

2 . The substrate of claim 1 , wherein the matrix further comprises a plurality of discrete reference regions, wherein each reference region comprises one or more reference labels, wherein the one or more reference labels can be transferred from the matrix into a tissue sample upon contacting the substrate with the tissue sample.

3 . (canceled)

4 . The substrate of claim 1 , wherein the matrix comprises a natural or synthetic polymer.

5 . (canceled)

6 . The substrate of claim 1 , wherein the one or more barcode labels are fluorescent or non-fluorescent.

7 . The substrate of claim 2 , wherein the one or more reference labels comprise one or more fluorescent dyes and/or a staining dye.

8 - 10 . (canceled)

9 . The substrate of claim 1 , wherein the one or more barcode labels comprise a molecular tag indicating spatial coordinates within the matrix.

10 - 13 . (canceled)

11 . The substrate of claim 1 , wherein each labeling region comprises a unique combination of the one or more barcode labels.

12 . The substrate of claim 2 , wherein the labeling regions and/or reference regions are prepared by spotting droplets of a solution comprising the one or more barcode labels onto the matrix.

13 . (canceled)

14 . The substrate of claim 1 , wherein the labeling regions have a distance of about 250 μm, about 220 μm, about 200 μm, about 190 μm, about 180 μm, about 170 μm, about 160 μm, about 150 μm, about 140 μm, about 130 μm, about 120 μm, about 110 μm, about 100 μm, or about 50 μm between the centers of the labeling regions.

15 . The substrate of claim 1 , wherein the labeling regions have a diameter of from 0.5 μm to 25 μm, from 1 μm to 200 μm, or from 50 μm to 200 μm.

16 . The substrate of claim 1 , wherein the labeling regions are arranged in a grid.

17 . The substrate of claim 1 , wherein the substrate comprises about 3,000, about 4,000, about 5,000, about 6,000, about 7,000, about 8,000, about 9,000, about 10,000, about 25,000, about 50,000, about 100,000, about 200,000, about 300,000, about 400,000, about 500,000, about 600,000, about 700,000, about 800,000, about 900,000, or about 1,000,000 unique labeling regions in a 10 mm by 10 mm area.

21 . (canceled)

22 . The substrate of claim 1 , wherein the support comprises glass or synthetic polymer.

23 . (canceled)

24 . A method of spatially labeling cells in a tissue sample, comprising contacting a tissue sample comprising a plurality of cells with the substrate of claim 1 for a time sufficient for at least a portion of the one or more barcode labels to transfer into at least a portion of the plurality of cells.

25 - 28 . (canceled)

29 . The method of claim 24 , wherein the method further comprises dissociating the tissue sample into individual cells after contacting the sample with the substrate.

30 . The method of claim 24 , wherein the tissue sample is sandwiched between a glass slide or plastic slide and the substrate during the contacting.

31 . A kit for spatially labeling cells in a multi-cellular sample, comprising the substrate of claim 1 .

32 . (canceled)

33 . A method of spatial gene expression profiling in a multi-cellular sample, comprising:
(a) contacting a tissue sample comprising a plurality of cells with the substrate of claim 2 for a time sufficient for at least a portion of the one or more barcode labels to transfer into at least a portion of the plurality of cells; (b) imaging the substrate to determine positions of the reference regions; (c) extracting nuclei from each cell; and (d) analyzing gene expression in each individual cell.

34 . The method of claim 33 , wherein the method comprises dissociating the tissue sample into individual cells after imaging the substrate.

35 . (canceled)

36 . The method of claim 33 , wherein the tissue sample is sandwiched between a glass slide or plastic slide and the substrate during the contacting.

37 . (canceled)

Assignees

• Univ Washington

Inventors

• Regier Mary
• Stevens Kelly Rose
• Srivatsan Sanjay
• Trapnell Cole

Classifications

• G01N33/50 Primary

  Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing (measuring or testing processes involving enzymes or microorganisms, compositions or test papers therefor; processes for forming such compositions, condition responsive control in microbiological or enzymological processes C12Q) · CPC title

• C12Q1/6816 Primary

  characterised by the detection means (C12Q1/6804 takes precedence) · CPC title

• G01N33/533

  with fluorescent label · CPC title

• C12Q1/6876

  Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes · CPC title

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title