# US 12,111,239 B2 · Methods and kits for isolating whole cells from frozen tissues for cell fingerprinting > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 12,111,239 B2 - Document ID: US12111239B2 - Status: Granted - Kind: B2 - Application number: 17/308,393 - Filed: 2021-05-05 - Published: 2021-11-11 - Granted: 2024-10-08 - Earliest claimed: 2020-05-06 - Assignees: Peri-Nuc Labs, LLC - Applicants: Peri-Nuc Labs LLC - Inventors: Tattym E. Shaiken - Web page: https://patentgrep.com/patent/US12111239B2 ## CPC - G01N 1/4055: {by solubility techniques} - B01L 3/021: {Pipettes, i.e. with only one conduit for withdrawing and redistributing liquids} - G01N 1/286: {involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising (microtomes G01N1/06; pulverising in general B02C; mixing in general B01F)} - G01N 1/4077: {by other techniques involving separation of suspended solids} - G01N 1/44: Sample treatment involving radiation, e.g. heat - G01N 2001/2873: {Cutting or cleaving} - 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 ## Abstract The present disclosure relates to methods for isolating whole cells from frozen tissue samples. The present disclosure further provides kits for obtaining isolated whole cells from a frozen tissue sample. One benefit to the methods and kits can be the rapid isolation of whole cells from frozen tissues upon thawing. Another benefit to the methods and kits disclosed herein can be the rapid and efficient extraction of isolated whole cells from stroma material in frozen tissues. Another benefit to the methods and kits disclosed herein can be the isolation of whole cells from frozen tissues suitable for subsequent biochemical analysis. ## Claims ### Claim 1 A method of isolating whole cells from a frozen tissue sample comprising: (a) providing a frozen tissue sample, wherein the frozen tissue sample contains whole cells and stroma material; (b) cutting the frozen tissue sample into two or more tissue pieces using a tissue sample cutter, wherein the tissue sample cutter has a plurality of cutting blades; (c) forming an extraction mixture by incubating the two or more tissue pieces in a volume of tissue sample solution (TSS) for an incubation period; (d) forming a whole cell sample by pipetting a first portion of the extraction mixture from the tissue sample solution into an extraction pipette, and then (e) pipetting a second portion of the extraction mixture from the extraction pipette into a washing solution; and (f) collecting isolated whole cells from the whole cell sample by flowing the extraction mixture through a first separation funnel into a collection vessel. ### Claim 2 (depends on 1) The method of claim 1, provided that stroma material is present in the whole cell sample, further comprising reducing an amount of stroma material in the whole cell sample by flowing the extraction mixture through a second separation funnel into a second collection vessel. ### Claim 3 (depends on 1) The method of claim 1, wherein the tissue sample is a tumor sample, or the whole cells are tumor cells. ### Claim 4 (depends on 1) The method of claim 1, wherein the tissue sample has a weight of from about 1 gram to about 5 grams, or wherein the two or more tissue pieces have a width of from about 2 mm to about 6 mm or wherein the incubation period is from about 3 minutes to about 5 minutes, or wherein incubating the tissue pieces includes shaking the extraction mixture during a portion of the incubation period; or wherein the pipetting is performed for a pipetting period of from about 1 minute to about 15 minutes. ### Claim 5 (depends on 2) The method of claim 2, including further reducing an amount of stroma material in the whole cell sample by flowing the washing solution from the first collection vessel through the first separation funnel or from a second separation funnel into a second collection vessel. ### Claim 6 (depends on 1) The method of claim 1, further comprising: (a) forming a pellet of whole cells by centrifuging the washing solution in the collection vessel; (b) removing the washing solution from the collection vessel; (c) forming a washed cell suspension by suspending the pellet in a volume of cell wash solution (CWS); (d) forming a washed cell pellet and a washed cell supernatant by centrifuging the washed cell suspension of step (c); and (e) forming a sample of isolated whole cells by removing the washed cell supernatant of step (d) from the washed cell pellet. ### Claim 7 (depends on 6) The method of claim 6, wherein the centrifuging is performed at a gravitational force of from about 80 g to about 120 g and for a centrifuge period of from about 3 minutes to about 7 minutes. ### Claim 8 (depends on 6) The method of claim 6, wherein the TSS includes from about 10 mM to about 40 mM Hepes, from about 0 mM to about 120 mM KCl, from 0 mM to about 150 mM NaCl, from 0 mM to about 10 mM MgCl2, from 0 μM to about 2 μM CaCl2), and from 0% to about 10% glycerol; or wherein the CWS includes from about 10 mM to about 40 mM Hepes, from 0 mM to about 120 mM KCl, from 0 mM to about 150 mM NaCl, from 0 mM to about 10 mM MgCl2, from 0 μM to about 2 μM CaCl2), and from 0% to about 10% glycerol. ### Claim 9 (depends on 1) The method of claim 1, wherein the TSS includes at least one protease inhibitor, at least one phosphatase inhibitor, at least one DNase inhibitor, at least one RNase inhibitor, or a combination thereof. ### Claim 10 (depends on 9) The method of claim 9, further comprising performing at least one biochemical analysis on the isolated whole cells. ### Claim 11 (depends on 10) The method of claim 10, wherein the at least one biochemical analysis includes protein analysis, phosphorylated protein analysis, nucleic acid analysis, or a combination thereof. ### Claim 12 (depends on 1) The method of claim 1, further comprising maintaining the frozen tissue at a temperature below 0 degrees Celsius before and during cutting, or at a temperature of about 0 degrees Celsius to about 4 degrees Celsius before and during cutting.