# US 11,993,809 B2 · Method for analyzing cell sample heterogeneity

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- Number: US 11,993,809 B2
- Document ID: US11993809B2
- Status: Granted
- Kind: B2
- Application number: 17/260,572
- Filed: 2019-07-17
- Published: 2022-05-26
- Granted: 2024-05-28
- Earliest claimed: 2018-07-18
- Assignees: Max-DelbrüCk-Centrum FüR Molekulare Medizin In Der Helmholtz-Gemeinschaft
- Applicants: Max-DelbrüCk-Centrum FüR Molekulare Medizin In Der Helmholtz-Gemeinschaft
- Inventors: Christin Sünkel; Nikolaos Karaiskos; Nikolaus Rajewsky
- Web page: https://patentgrep.com/patent/US11993809B2

## CPC

- C12Q 1/6841: In situ hybridisation
- G01N 1/286: {involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising (microtomes G01N1/06; pulverising in general B02C; mixing in general B01F)}
- C12N 15/1065: {Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags}
- C12N 15/1096: {cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR}
- C12Q 1/6876: Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q 2600/16: Primer sets for multiplex assays
- G01N 2001/282: {with mapping; Identification of areas; Spatial correlated pattern}
- G01N 2001/2873: {Cutting or cleaving}
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention relates to a method for analyzing a biological sample on a single cell level by compartmentalizing said sample using a grid and performing an optimized combinatorial indexing protocol within cells of the compartmentalized tissue while deducing cell-specific information regarding the cell identity and activity in the spatial context within the sample.

## Claims

### Claim 1

A method for analyzing a tissue sample comprising cells, wherein said method comprises the following steps:
(a) compartmentalizing said sample with a grid into at least two compartments, wherein the grid comprises at least two break-through holes which define said at least two compartments of the sample and wherein at least one of said at least two compartments of the sample comprises at least one cell containing a plurality of molecules;
(b) labeling the plurality of molecules within said at least one cell in situ with at least a compartment-specific label,
wherein before step (a) the sample is fixed and permeabilized, and wherein steps (a) and (b) are performed repeatedly and alternating, wherein before every repetition of step (a) at least one of the following steps is performed:
rotating the grid in relation to the surface defined by the two largest dimensions of the sample around an axis perpendicular to said surface;
rotating the grid in relation to said surface around an axis parallel to said surface;
shifting the grid in relation and parallel to said surface;
exchanging the grid by a further grid, wherein said further grid comprises at least two break-through holes which differ with respect to their number and/or at least one cross-sectional size and/or at least one cross-sectional geometry from the at least two break-through holes of any of the at least one grid applied in any of the previous steps (a);
choosing one of the at least one grid applied in any of the previous steps (a);
(c) obtaining a single cell suspension from the compartmentalized sample comprising the compartment-specific labeled molecules within the cell(s);
(d) mixing at least a fraction of said single cell suspension and distributing it over at least two vessels;
(e) labeling within the at least two vessels the plurality of molecules within the cell(s) with a vessel-specific label; and
(f) obtaining data for at least a fraction of the sequence of at least a fraction of the plurality of molecules, said data being obtained using a sequencing or genotyping method or a next generation sequencing method.

### Claim 2 (depends on 1)

The method according to claim 1, wherein at least two grids with at least two different grid geometries are applied in at least three successively performed rounds of compartmentalization and compartment-specific labeling.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the tissue sample is a cryosection of a tissue.

### Claim 4 (depends on 3)

The method according to claim 3, wherein the cryosection has a slice thickness between 1 μm and 50 mm or between 5 μm and 15 μm.

### Claim 5 (depends on 1)

The method according to claim 1, the method further comprising the following steps:
(i) obtaining a first suspension comprising at least a fraction of the at least one cell containing a plurality of molecules derived from step (b) and distributing at least a fraction of the first suspension over a first set of at least two vessels; and
(ii) labeling the plurality of molecules comprised in the suspensions contained in the first set of at least two vessels with a first vessel-specific label within said at least one cell.

### Claim 6 (depends on 5)

The method according to claim 5, the method further comprising the following steps:
(iii) obtaining a further suspension, comprising at least a fraction of the at least one cell containing a plurality of molecules, by mixing at least a fraction of the suspensions derived
from step (ii) in case step (iii) is performed for the first time, or
from step (iv) in case steps (iii) and (iv) have been performed at least once,
and distributing at least a fraction of the further suspension over a further set of at least two vessels; and
(iv) labeling the plurality of molecules comprised in the suspensions contained in the further set of at least two vessels with at least a further vessel-specific label.

### Claim 7 (depends on 6)

The method according to claim 6, wherein lysis of the at least one cell is performed
after step (b), or
in case step (iii) and step (iv) are performed once: after step (iii) and before step (iv), or
in case step (iii) and step (iv) are performed in multiple iterations: after step (iii) and before step (iv) of the last iteration.

### Claim 8 (depends on 1)

The method according to claim 1, wherein the method further comprises identifying compartment-specific and/or vessel-specific labels, obtaining cell-specific parameters, and obtaining information of relative spatial cell positions.

### Claim 9 (depends on 1)

The method according to claim 1, wherein the method further comprises comparing results for said sample to results obtained for at least a second sample and/or information obtained by other methods at least for said sample.

### Claim 10 (depends on 1)

The method according to claim 1, wherein the method further comprises obtaining and/or analyzing at least one biomarker indicative for a condition.

### Claim 11 (depends on 1)

The method according to claim 1, wherein before step (a) the sample is fixed and permeabilized using methanol.

### Claim 12 (depends on 1)

The method according to claim 1, wherein the method further comprises positioning the sample on a base of a grid system, positioning the grid on the surface of the sample that is opposite to the surface of the sample that is in contact with the base, and moving the grid towards the base until the grid is in contact with the base.

### Claim 13 (depends on 1)

The method according to claim 1, wherein said molecules are naturally occurring, synthetic and/or engineered molecules comprising nucleic acids and/or polypeptides.

### Claim 14 (depends on 13)

The method according to claim 13, wherein each of the compartment-specific labels has a compartment-specific nucleotide sequence of 1 nucleotide to 200 nucleotides in length and/or wherein each of the vessel-specific labels has a vessel-specific nucleotide sequence of 1 nucleotides to 200 nucleotides in length.

### Claim 15 (depends on 14)

The method according to claim 14, wherein each of the compartment-specific labels has a compartment-specific nucleotide sequence of 5 nucleotides to 25 nucleotides in length and/or wherein each of the vessel-specific labels has a vessel-specific nucleotide sequence of 5 nucleotides to 25 nucleotides in length.

### Claim 16 (depends on 1)

The method according to claim 1, wherein step (b) comprises reverse transcribing the plurality of molecules within the at least one cell using compartment-specific labeled primers.

### Claim 17 (depends on 1)

The method according to claim 1, wherein each compartment does not comprise more than one cell.

### Claim 18 (depends on 1)

The method according to claim 1, wherein the plurality of molecules comprising at least one compartment-specific label is labeled with at least one vessel-specific label by ligation.

### Claim 19 (depends on 1)

The method according to claim 1, wherein the plurality of molecules comprising at least one compartment-specific label is amplified by PCR.

### Claim 20 (depends on 19)

The method according to claim 19, wherein at least one further vessel-specific label is added to the molecules by PCR.

### Claim 21 (depends on 1)

The method according to claim 1, further comprising the steps of performing second strand synthesis of the plurality of molecules and/or tagmentation of the plurality of molecules.