# US 10,370,649 B2 · Device and method for cell nuclei preparation > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,370,649 B2 - Document ID: US10370649B2 - Status: Granted - Kind: B2 - Application number: 15/317,039 - Filed: 2015-06-17 - Published: 2017-05-04 - Granted: 2019-08-06 - Earliest claimed: 2014-06-30 - Assignees: Ge Healthcare UK Limited - Applicants: GE Healthcare UK Limited - Inventors: Michael Kenneth Kenrick; Geraint Seymour - Web page: https://patentgrep.com/patent/US10370649B2 ## CPC - C12N 15/1006: {by means of a solid support carrier, e.g. particles, polymers} - B01L 3/502: {with fluid transport, e.g. in multi-compartment structures} - C12N 15/1017: {by filtration, e.g. using filters, frits, membranes} - B01L 2200/0631: Purification arrangements, e.g. solid phase extraction [SPE] - B01L 2200/16: Reagents, handling or storing thereof - B01L 2300/044: pierceable, e.g. films, membranes - B01L 2300/0681: Filter - B01L 2300/12: Specific details about materials - B01L 2400/0478: pistons - B01L 2400/049: vacuum - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE ## Abstract The present invention relates to the field of nucleic acid collection and storage, and in particular to the collection and long-term storage of nuclei. The invention provides a device and method which can be used to capture and store nuclei at ambient temperatures allowing the subsequent isolation of nucleic acids by passive washing with a wash buffer. The present invention also provides a kit including the device of the invention suitable for carrying out the method of the invention. ## Claims ### Claim 1 A device comprising: (i) a container comprising an open upper end, a sealed lower end and a chamber therebetween defined by chamber walls; wherein said container further comprises a hollow tube adjacent the upper end allowing gaseous and liquid communication with the chamber and a removable base sealing the lower end, and wherein said chamber contains a cell lysis reagent; (ii) a plunger comprising a hollow plunger body defined by plunger walls wherein said plunger walls have a shape conforming to and smaller than said chamber to allow a bottom end of the plunger to sealably slide within the chamber against the chamber walls, a septum cap sealing a top end of said plunger body, and a solid support spanning an interior transverse section of said plunger body, wherein said solid support is a permeable membrane; and (iii) a vacuum port connected to the hollow tube, wherein negative pressure is created within the chamber through the hollow tube attached to the vacuum port. ### Claim 2 (depends on 1) The device as defined in claim 1, wherein said lysis reagent is a red cell lysis reagent. ### Claim 3 (depends on 2) The device as defined in claim 2, wherein said red cell lysis reagent is a sucrose-triton buffer. ### Claim 4 (depends on 1) The device as defined in claim 1, wherein said lysis reagent is water. ### Claim 5 (depends on 1) The device as defined in claim 1, wherein said lysis reagent is an anionic surfactant. ### Claim 6 (depends on 5) The device as defined in claim 5, wherein said chamber further contains a collection buffer. ### Claim 7 (depends on 1) The device as defined in claim 1, wherein said solid support is a permeable membrane selected from the group consisting of polyester membrane, polyamide membrane, polycarbonate membrane and cellulose membrane. ### Claim 8 (depends on 7) The device as defined in claim 7, wherein said solid support is a polyester membrane. ### Claim 9 (depends on 8) The device as defined in claim 8, wherein said solid support is located at the bottom end of said plunger body. ### Claim 10 A method comprising: (i) introducing a cellular sample into a container wherein said container has an open upper end and sealed lower end and a chamber therebetween defined by chamber walls, wherein said container further comprises a hollow tube adjacent the upper end allowing gaseous and liquid communication with the chamber, wherein negative pressure is created within the chamber through a vacuum port attached to the hollow tube, wherein said container contains a collection buffer comprising a lysis reagent at a concentration sufficient to selectively lyse the cytoplasmic membranes and a small proportion of the nuclear membranes present in the cellular sample and leave a large proportion of the cell nuclei intact; (ii) inserting a plunger into the upper end of said container wherein said plunger comprises a hollow plunger body defined by plunger walls, wherein said walls have a shape conforming to and smaller than said chamber, a septum cap sealing the top end of said plunger body, and a solid support comprising a permeable membrane spanning an interior transverse section of said plunger body; (iii) pressing said plunger towards said lower end of said container so that said solid support passes through said cellular sample and the cell nuclei and other cellular components bind to the solid support leaving filtrate behind; and, (iv) removing said filtrate. ### Claim 11 (depends on 10) The method as defined in claim 10, wherein said lysis reagent is a red cell lysis reagent. ### Claim 12 (depends on 11) The method as defined in claim 11, wherein said red cell lysis reagent is a sucrose-triton buffer. ### Claim 13 (depends on 10) The method as defined in claim 10, wherein said lysis reagent is water. ### Claim 14 (depends on 10) The method as defined in claim 10, wherein said lysis reagent is an anionic surfactant. ### Claim 15 (depends on 14) The method as defined in claim 14, wherein said cellular sample has a volume greater than 1 ml. ### Claim 16 (depends on 15) The method as defined in claim 15, wherein said cellular sample has a volume greater than 5 ml. ### Claim 17 (depends on 16) The method as defined in claim 16, wherein said cellular sample has a volume greater than 10 ml. ### Claim 18 (depends on 17) The method as defined in claim 17, wherein said intact cell nuclei contain nucleic acid. ### Claim 19 (depends on 18) The method as defined in claim 18, wherein said nucleic acid comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). ### Claim 20 (depends on 19) The method as defined in claim 19, which further comprises the step (v) of storing the intact nuclei on the solid support at ambient temperature. ### Claim 21 (depends on 20) The method as defined in claim 20, comprising the additional step of submerging the solid support in an organic solvent after said removing step (iv) and before said storing step (v). ### Claim 22 (depends on 21) The method as defined in claim 21, wherein said organic solvent is an alcohol. ### Claim 23 (depends on 22) The method as defined in claim 22, wherein said alcohol is ethanol or isopropanol. ### Claim 24 (depends on 23) The method as defined in claim 23, wherein said solid support is air dried prior to said storing step (v). ### Claim 25 (depends on 10) The method as defined in claim 10, wherein said solid support is a permeable membrane selected from the group consisting of polyester membrane, polyamide membrane, polycarbonate membrane and cellulose membrane. ### Claim 26 (depends on 25) The method as defined in claim 25, wherein said solid support is a polyester membrane. ### Claim 27 (depends on 26) The method as defined in claim 26, which comprises the additional step (vi) of recovering a nucleic acid from the intact nuclei. ### Claim 28 (depends on 27) The method as defined in claim 27, wherein said recovering step (vi) comprises lysing the intact nuclei on the solid support. ### Claim 29 (depends on 28) The method as defined in claim 28, wherein said recovering step (vi) comprises centrifugation. ### Claim 30 (depends on 29) The method as defined in claim 29, wherein said recovering step is preceded by passive washing said solid support. ### Claim 31 (depends on 30) The method as defined in claim 30, wherein said cellular sample is introduced into said container directly from a subject. ### Claim 32 (depends on 1, 10) A kit comprising the device as defined in claim 1 and instructions for carrying out the method of claim 10.