# US 10,246,683 B2 · Method and assembly for extraction of regenerative cellular components from adipose tissue > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,246,683 B2 - Document ID: US10246683B2 - Status: Granted - Kind: B2 - Application number: 15/329,792 - Filed: 2014-07-31 - Published: 2017-07-13 - Granted: 2019-04-02 - Earliest claimed: 2014-07-31 - Assignees: MyStem Limited - Applicants: MyStem Limited - Inventors: Piergiuseppe Ivona - Web page: https://patentgrep.com/patent/US10246683B2 ## CPC - C12N 5/0667: {Adipose-derived stem cells [ADSC]; Adipose stromal stem cells} - A61K 35/35: Fat tissue; Adipocytes; Stromal cells; Connective tissues (adipose-derived stem cells A61K35/28; collagen A61K38/39) - C12M 27/10: {Rotating vessel} - C12M 29/04: {Filters; Permeable or porous membranes or plates, e.g. dialysis} - C12M 45/02: {by mechanical forces; Stirring; Trituration; Comminuting (crushing, pulverizing, disintegrating in general B02C)} - C12M 47/04: {Cell isolation or sorting (purging biological preparations of unwanted cells C12N5/0081, determining the presence or kind of microorganism C12Q1/04)} - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - 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} ## Abstract The present invention concerns a novel method and assembly for extraction of regenerative cellular components from an adipose tissue, the method comprising the steps of filtering a harvested adipose tissue for recovering its liquid fraction by means of a filtering element having a first cut-off value comprised between 40 microns and 70 microns; separating regenerative cellular components present in the liquid fraction by means of cell size segregation which blocks cells bigger than a second cut-off value ranging from 8 to 20 microns; and recovering the regenerative cells-rich fraction comprising the regenerative cellular components not having being blocked during the separating step. ## Claims ### Claim 1 A method for extracting regenerative cellular components from adipose tissue comprising the steps of: a) filtering a harvested adipose tissue for recovering its liquid fraction by means of a filtering element having a first cut-off value between 40 microns and 70 microns; b) separating regenerative cellular components present in the liquid fraction by means of cell size segregation, which blocks cells bigger than a second cut-off value ranging from 8 to 20 microns; and c) recovering the liquid regenerative cells-rich fraction comprising the regenerative cellular components not having being blocked during the separating step b); wherein steps a)-c) are carried out in a time ranging from 1 to 5 minutes. ### Claim 2 (depends on 1) The method according to claim 1, wherein the first cut-off value is between 45 microns and 60 microns. ### Claim 3 (depends on 1) The method according to claim 1, wherein the second cut-off value is between 10 microns and 18 microns. ### Claim 4 (depends on 1) The method according to claim 1, wherein step b) of separation of regenerative cellular components present in the liquid fraction is performed by filtration or sedimentation techniques or by means of a microfluidic array. ### Claim 5 (depends on 1) The method according to claim 1, wherein the recovered regenerative cellular components are a stromal vascular fraction of the adipose tissue. ### Claim 6 A method for extracting regenerative cellular components from adipose tissue comprising the steps of: a) filtering a harvested adipose tissue for recovering its liquid fraction by means of a filtering element having a first cut-off value between 40 microns and 70 microns; b) separating regenerative cellular components present in the liquid fraction by means of cell size segregation, which blocks cells bigger than a second cut-off value ranging from 8 to 20 microns; c) recovering the liquid regenerative cells-rich fraction comprising the regenerative cellular components not having being blocked during the separating step b); and d) separating regenerative cellular components present in a dense fraction filtered from the liquid fraction in step a). ### Claim 7 (depends on 6) The method according to claim 6, wherein the separation of regenerative cellular component present in the dense fraction of step d) comprises: recovering a dense fraction of adipose tissue filtered from the liquid fraction in step a); adding saline solution to the recovered dense fraction of adipose tissue; subjecting the mixture of saline solution and dense fraction of adipose tissue to a homogenization step for reducing the dimension of adipocytes comprised in the dense fraction; centrifuging the homogenized mixture until phase separation and precipitation of the homogenized mixture takes place; and after centrifugation recovering a pellet deriving from the phase separation and precipitation of the homogenized mixture. ### Claim 8 (depends on 7) The method according to claim 7, wherein the homogenization step for reducing the dimensions of adipocytes comprises subjecting the mixture of the dense fraction of adipose tissue and saline solution to micronization. ### Claim 9 (depends on 8) The method according to claim 8, wherein the micronization step comprises: injecting a recovered first quantity of the dense fraction of adipose tissue into a micronizer together with saline solution in the same first quantity; micronizing for a first preset time period; extracting the micronized mixture of the dense fraction of adipose tissue and saline solution from the micronizer. ### Claim 10 (depends on 9) The method according to claim 9, wherein the first preset time period is between 3 minutes and 30 seconds. ### Claim 11 (depends on 9) The method according to claim 9, wherein the centrifuging step has a duration between 10 minutes and 3 minutes. ### Claim 12 A method for extracting regenerative cellular components from adipose tissue comprising the steps of: a) filtering a harvested adipose tissue for recovering its liquid fraction by means of a filtering element having a first cut-off value between 40 microns and 70 microns; b) separating regenerative cellular components present in the liquid fraction by means of cell size segregation, which blocks cells bigger than a second cut-off value ranging from 8 to 20 microns; and c) recovering the liquid regenerative cells-rich fraction comprising the regenerative cellular components not having being blocked during the separating step b); wherein steps a)-c) are carried out at room temperature. ### Claim 13 An apparatus for extraction of regenerative cellular components from an adipose tissue comprising: a filtering element in a filtering pouch having a first inlet connector and a first outlet connector, the first inlet and first outlet connectors being air-contamination free, the filtering element having a first cut-off value between 40 microns and 70 microns for separating the liquid fraction from the dense fraction of a harvested adipose tissue; a separation device having a second cut-off value ranging from 8 to 20 microns; and a recovering chamber for collecting the regenerative cells-rich fraction comprising the regenerative cellular components not having being blocked by the separation device. ### Claim 14 (depends on 13) The apparatus according to claim 13, wherein the first inlet connector and/or the first outlet connector are needle free connectors. ### Claim 15 (depends on 13) The apparatus according to claim 13, wherein the filtering pouch comprises an air inlet with an air filter. ### Claim 16 (depends on 13) The apparatus according to claim 13, wherein the separation device comprises a second inlet connector and a second outlet connector, the second inlet and second outlet connectors being air-contamination free. ### Claim 17 (depends on 16) The apparatus according to claim 16, wherein the first outlet connector of the filtering pouch in the filtering element is connected to said second inlet connector of the separation device by interposition of an one-way valve. ### Claim 18 (depends on 13) The apparatus according to claim 13 further comprising a micronizing device and a centrifuging device. ### Claim 19 (depends on 2) The method according to claim 2, wherein the first cut-off value is between 48 microns and 55 microns. ### Claim 20 (depends on 3) The method according to claim 3, wherein the second cut-off value is between 13 microns and 16 microns. ### Claim 21 (depends on 10) The method according to claim 10, wherein the first preset time period is about 1 minute. ### Claim 22 (depends on 11) The method according to claim 11, wherein the centrifuging step has a duration equal to about 5 minutes. ### Claim 23 (depends on 6) The method according to claim 6, wherein the second cut-off value is between 10 microns and 18 microns. ### Claim 24 (depends on 6) The method according to claim 6, wherein step b) of separation of regenerative cellular components present in the liquid fraction is performed by filtration or sedimentation techniques or by means of a microfluidic array. ### Claim 25 (depends on 6) The method according to claim 6, wherein the recovered regenerative cellular components are a stromal vascular fraction of the adipose tissue. ### Claim 26 (depends on 12) The method according to claim 12, wherein the second cut-off value is between 10 microns and 18 microns. ### Claim 27 (depends on 12) The method according to claim 12, wherein step b) of separation of regenerative cellular components present in the liquid fraction is performed by filtration or sedimentation techniques or by means of a microfluidic array. ### Claim 28 (depends on 12) The method according to claim 12, wherein the recovered regenerative cellular components are a stromal vascular fraction of the adipose tissue.