# US 10,731,132 B2 · Methods and apparatuses for umbilical cord blood collection and isolation of cells > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,731,132 B2 - Document ID: US10731132B2 - Status: Granted - Kind: B2 - Application number: 14/776,655 - Filed: 2014-03-14 - Published: 2016-03-10 - Granted: 2020-08-04 - Earliest claimed: 2013-03-15 - Assignees: Amniotics AB - Applicants: Amniotics AB - Inventors: Marcus Kare Torleif Larsson; Andreas Nils Walter Herbst; Niels-bjarne Roland Woods - Web page: https://patentgrep.com/patent/US10731132B2 ## CPC - C12N 5/0665: {Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood} - C12N 5/0087: {Purging against subsets of blood cells, e.g. purging alloreactive T cells} - C12N 5/0647: {Haematopoietic stem cells; Uncommitted or multipotent progenitors} - C12N 5/069: {Vascular Endothelial cells} - C12N 5/0692: {Stem cells; Progenitor cells; Precursor cells} - C12N 2500/02: Atmosphere, e.g. low oxygen conditions - C12N 2506/11: from blood or immune system cells - C12N 2523/00: Culture process characterised by temperature - C12N 2533/54: Collagen; Gelatin - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract The invention relates to method for the separation of cord blood endothelial cells from hematopoietic cells in a manner that preserves the hematopoietic cells' repopulating ability and primitivity, without significantly reducing the yield of hematopoietic cells. The said endothelial cells from cord blood have a utility in cell based therapeutics. The system described takes advantage of the endogenous adherence properties of the cells to facilitate the separation and separate storage of the cell types. ## Claims ### Claim 1 A method for cell separation, comprising: plating a freshly harvested umbilical cord blood sample that has not been enriched, stored, thawed, or washed on a surface at a surface density of approximately 100 million cells per 100 cm2, the surface coated in a component configured to selectively bind with endothelial cells within the whole umbilical cord blood sample; allowing adhesion of cells in said umbilical cord blood sample to said surface for about 5 minutes to about 60 minutes, to generate an adherent fraction and a non-adherent fraction of said whole umbilical cord blood sample; separating said non-adherent fraction from said adherent fraction, wherein said non-adherent fraction comprises a hematogenic progenitor cell or a hematopoietic stem cell; wherein said adherent fraction comprises at least one of an endothelial cell and an endothelial progenitor cell; and processing and storing the non-adherent fraction. ### Claim 2 (depends on 1) The method of claim 1, wherein separating said non-adherent fraction from said adherent fraction does not cause detachment of the majority of the cells in said adherent fraction from the surface. ### Claim 3 (depends on 1) The method of claim 1, wherein about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% of cells in said adherent fraction remain attached to the surface following separating said non-adherent fraction from said adherent fraction. ### Claim 4 (depends on 1) The method of claim 1, wherein said umbilical cord blood sample is from a human. ### Claim 5 (depends on 1) The method of claim 1, wherein said umbilical cord blood sample is frozen and thawed before the plating step. ### Claim 6 (depends on 1) The method of claim 1, wherein the said umbilical cord blood sample is plated at a temperature between about 0° C. and about 37° C. ### Claim 7 (depends on 1) The method of claim 1, wherein the surface is coated with an extracellular matrix component. ### Claim 8 (depends on 7) The method of claim 7, wherein the extracellular matrix component is collagen I, gelatin, or a combination thereof. ### Claim 9 (depends on 1) The method of claim 1, wherein the cell viability, primitivity, proliferation ability, differentiation ability, and/or repopulating ability of the hematogenic progenitor cell or hematopoietic stem cell is maintained. ### Claim 10 (depends on 1) The method of claim 1, wherein the cell viability and/or primitivity of cells in the adherent fraction is maintained. ### Claim 11 (depends on 1) The method of claim 1, further comprising isolating an adherent cell from the adherent fraction and reprogramming the isolated adherent cell to a stem cell or progenitor cell. ### Claim 12 (depends on 1) The method of claim 1, further comprising expanding the hematogenic progenitor cell or hematopoietic stem cell. ### Claim 13 (depends on 12) The method of claim 12, further comprising: (1) isolating a more primitive cell from the expanded hematogenic progenitor cell or hematopoietic stem cell population, or (2) reprogramming said hematogenic progenitor cell or hematopoietic stem cell to a more primitive cell. ### Claim 14 (depends on 1) The method of claim 1, further comprising isolating an adherent cell from the adherent fraction, and: (a) reprogramming the isolated adherent cell to a stem cell or progenitor cell, and differentiating said stem cell or progenitor cell to a somatic cell; or (b) reprogramming the isolated adherent cell to a somatic cell directly. ### Claim 15 (depends on 1) The method of claim 1, wherein a device is used for cell separation from the umbilical cord blood sample, the device comprising a first vessel, a second vessel, and a sterile tubing with stopcock flow regulation for the transfer of cells in suspension between the vessels, and wherein one of the vessels is pre-coated for cell attachment of cells in the adherent fraction, and the other vessel is a collection vessel for cells in the non-adherent fraction. ### Claim 16 (depends on 15) The method of claim 15, wherein the device is prefilled with a hypoxic, normoxic, or hyperoxic gas. ### Claim 17 (depends on 1) The method of claim 1, wherein said adherent fraction comprises a mesenchymal stem cell. ### Claim 18 (depends on 17) The method of claim 17, further comprising isolating said endothelial cell, endothelial progenitor cell, and/or mesenchymal stem cell from said adherent fraction. ### Claim 19 (depends on 1) The method of claim 1, further comprising isolating said hematogenic progenitor cell or hematopoietic stem cell from said non-adherent fraction. ### Claim 20 (depends on 1) The method of claim 1, further comprising using the non-adherent fraction for treatment of a hematologic disorder.