# US 11,441,976 B2 · Method and apparatus for processing tissue samples

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- Number: US 11,441,976 B2
- Document ID: US11441976B2
- Status: Granted
- Kind: B2
- Application number: 17/513,204
- Filed: 2021-10-28
- Published: 2022-03-17
- Granted: 2022-09-13
- Earliest claimed: 2016-11-29
- Assignees: S2 Genomics, Inc.
- Applicants: S2 Genomics, Inc.
- Inventors: Stevan Bogdan Jovanovich; Frank Zaugg; Kaiwan Chear; Roger McIntosh; Nathan Pereira
- Web page: https://patentgrep.com/patent/US11441976B2

## CPC

- G01N 1/31: Apparatus therefor
- G01N 35/026: {having blocks or racks of reaction cells or cuvettes}
- B01L 3/502: {with fluid transport, e.g. in multi-compartment structures}
- B01L 7/52: {with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples}
- C12M 47/04: {Cell isolation or sorting (purging biological preparations of unwanted cells C12N5/0081, determining the presence or kind of microorganism C12Q1/04)}
- C12M 47/06: {Hydrolysis; Cell lysis; Extraction of intracellular or cell wall material (lysis of microorganisms C12N1/06; extracting or separating nucleic acids from biological samples C12N15/1003)}
- C12N 1/06: Lysis of microorganisms
- C12N 1/066: by physical processes
- C12N 15/1003: {Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor}
- C12Q 1/6851: Quantitative amplification
- G01N 1/286: {involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising (microtomes G01N1/06; pulverising in general B02C; mixing in general B01F)}
- G01N 35/00: Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
- B01L 2200/0647: Handling flowable solids, e.g. microscopic beads, cells, particles
- B01L 2300/021: Identification, e.g. bar codes
- B01L 2300/0681: Filter
- B01L 2300/1822: using Peltier elements
- B01L 2300/1827: using resistive heater
- B01L 2400/043: magnetic forces
- B01L 2400/0478: pistons
- G01N 2001/2866: {Grinding or homogeneising}
- G01N 2035/00198: {Dissolution analysers}
- G01N 2035/00346: {Heating or cooling arrangements}
- G01N 2035/00564: {Handling or washing solid phase elements, e.g. beads}
- G01N 2035/00752: {bar codes}
- G01N 2035/0436: {with pre-packaged reagents, i.e. test-packs}
- G01N 2035/1058: {for mixing}
- 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)
- B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- C12M: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; {APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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

## Abstract

A system, methods, and apparatus are described to collect and prepare single cells, nuclei, subcellular components, and biomolecules from specimens including tissues. The system can perform enzymatic and/or physical disruption of the tissue to dissociate it into single-cells or nuclei in suspension or subcellular components including nucleic acids. In some embodiments, the titer of dissociated cells is monitored at intervals and the viability determined. In some embodiments, the processing is adjusted according to the measurements of the titer and viability. In some embodiments, the single-cells or nuclei in suspension are washed and resuspended in the buffer or media of choice. In some embodiments, the conditions are chosen to produce nuclei. In other embodiments, the single-cells or nuclei are purified by affinity paramagnetic bead processing. In some embodiments, matched bulk nucleic acid to the single-cells is produced. In other embodiments, single-cell libraries, or nuclei libraries, or matched bulk libraries, or bulk libraries are produced. The single cells or nuclei can then be further processed by FACS, DNA sequencing, mass spectrometry, fluorescence, or other methods. In other embodiments, the tissue processing is integrated with an analytical system to produce a sample-to-answer system such as a tissue-to-genomics system.

## Claims

### Claim 1

A method of analyzing subcellular organelles comprising:
(a) providing a tissue sample or cell suspension sample;
(b) performing mechanical or chemical disruption of the tissue or cell suspension sample in a chamber situated in a cartridge, while the cartridge is engaged with a cartridge interface of an instrument, to produce debris and released subcellular organelles in the chamber;
(c) separating a plurality of the released subcellular organelles from a portion of the debris into a liquid suspension;
(d) collecting the plurality of the released subcellular organelles in the liquid suspension; and
(e) analyzing individual subcellular organelles from the plurality of released subcellular organelles.

### Claim 2 (depends on 1)

The method of claim 1, wherein the providing a tissue sample or cell suspension sample comprises providing a tissue sample.

### Claim 3 (depends on 2)

The method of claim 2, wherein the tissue sample comprises an organ or organ fragment.

### Claim 4 (depends on 3)

The method of claim 3, wherein the organ or organ fragment is from one or more organs selected from the group consisting of: kidney, lung, spleen, liver, gut, and brain.

### Claim 5 (depends on 2)

The method of claim 2, wherein the tissue sample is an organ or organ fragment that is 100 mg or less.

### Claim 6 (depends on 5)

The method of claim 5, further comprising collecting at least 75,000 intact released subcellular organelles per mg of the organ or organ fragment.

### Claim 7 (depends on 6)

The method of claim 6, wherein the organ or organ fragment is from kidney, lung, spleen, liver, brain or gut.

### Claim 8 (depends on 6)

The method of claim 6, wherein the at least 75,000 intact released subcellular organelles per mg of the organ or organ fragment comprise at least 75,000 intact released nuclei per mg of the organ or organ fragment.

### Claim 9 (depends on 1)

The method of claim 1, wherein the separating of the released subcellular organelles from a portion of the debris is automated.

### Claim 10 (depends on 6)

The method of claim 6, wherein the at least 75,000 intact released subcellular organelles per mg of the organ or organ fragment comprise at least 75,000 intact released ribosomes or mitochondria, or combination thereof, per mg of the organ or organ fragment.

### Claim 11 (depends on 1)

The method of claim 1, further comprising contacting the tissue sample with a detergent, surfactant, or enzyme prior to, during, or following the mechanical or chemical disruption of the tissue sample in the apparatus.

### Claim 12 (depends on 2)

The method of claim 2, wherein the tissue sample is an organ fragment that is 25 mg or less.

### Claim 13 (depends on 2)

The method of claim 2, wherein the tissue sample comprises tumor tissue.

### Claim 14 (depends on 2)

The method of claim 2, wherein the tissue sample comprises frozen tissue.

### Claim 15 (depends on 2)

The method of claim 2, wherein the tissue sample comprises fixed tissue.

### Claim 16 (depends on 2)

The method of claim 2, wherein the performing mechanical or chemical disruption of the tissue sample comprises automatically performing mechanical disruption of the tissue sample.

### Claim 17 (depends on 1)

The method of claim 1, wherein the tissue sample or cell suspension sample comprises a cell suspension comprising tumor cells.

### Claim 18 (depends on 1)

The method of claim 1, wherein the collecting the plurality of the released subcellular organelles in the liquid suspension comprises collecting individual nuclei in the liquid suspension.

### Claim 19 (depends on 1)

The method of claim 1, wherein the collecting the plurality of the released subcellular organelles in the liquid suspension comprises collecting ribosomes or mitochondria in the liquid suspension.

### Claim 20 (depends on 1)

The method of claim 1, wherein the collecting the plurality of the released subcellular organelles in the liquid suspension comprises collecting at least 75,000 released intact nuclei per mg of the tissue sample.

### Claim 21 (depends on 1)

The method of claim 1, wherein the collecting the plurality of the released subcellular organelles in the liquid suspension comprises collecting between 75,000 to 1,600,000 nuclei per mg of the tissue sample.

### Claim 22 (depends on 1)

The method of claim 1, wherein the collecting the plurality of the released subcellular organelles in the liquid suspension comprises automatically moving the released subcellular organelles in the liquid suspension into a strain chamber comprising a strainer or filter.

### Claim 23 (depends on 1)

The method of claim 1, wherein the cartridge is disposable.

### Claim 24 (depends on 1)

The method of claim 1, wherein analyzing the released subcellular organelles comprises performing optical imaging of the released subcellular organelles.

### Claim 25 (depends on 1)

The method of claim 1, wherein analyzing the released subcellular organelles comprises performing sequencing of DNA or RNA derived from the released subcellular organelles.

### Claim 26 (depends on 6)

The method of claim 6, wherein analyzing individual subcellular organelles from the plurality of released subcellular organelles comprises performing optical imaging to measure titer, clumping, viability, or a combination thereof of the released subcellular organelles.

### Claim 27 (depends on 8)

The method of claim 8, wherein analyzing individual subcellular organelles from the plurality of released subcellular organelles comprises performing sequencing of DNA or RNA derived from a portion of the at least 75,000 intact released nuclei per mg of the organ or organ fragment.

### Claim 28

A sample processing method comprising:
(a) providing a tissue sample, wherein the tissue sample is an organ fragment that is 20 mg or less;
(b) loading the tissue sample into a chamber situated in a cartridge;
(c) prior to, or following (b), engaging the cartridge with a cartridge interface of an instrument;
(d) performing mechanical disruption of the sample in the chamber to produce material comprising debris and released cells;
(e) separating the released cells from a portion of the debris; and
(f) collecting at least 20,000 viable released cells per mg of the tissue sample.

### Claim 29 (depends on 28)

The method of claim 28, wherein the organ fragment is a kidney, lung, spleen, liver, or gut fragment.

### Claim 30 (depends on 28)

The method of claim 28, further comprising analyzing the released cells.