# US 2019/0169573 A1 · Cells derived from adipose tissue and methods of isolating and using the same > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2019/0169573 A1 - Document ID: US20190169573A1 - Status: Published application - Kind: A1 - Application number: 16/273,059 - Filed: 2019-02-11 - Published: 2019-06-06 - Earliest claimed: 2002-03-19 - Assignees: Keith Leonard March - Inventors: Keith Leonard March; Jalees Rehman - Web page: https://patentgrep.com/patent/US20190169573A1 ## CPC - C12N 5/0667: {Adipose-derived stem cells [ADSC]; Adipose stromal stem cells} - A61P 9/00: Drugs for disorders of the cardiovascular system - C12N 5/0619: {Neurons} - C12N 5/0653: {Adipocytes; Adipose tissue} - A61K 35/12: Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells (vaccines or medicinal preparations containing antigens or antibodies A61K39/00) - C12N 2501/105: Insulin-like growth factors [IGF] - C12N 2501/11: Epidermal growth factor [EGF] - C12N 2501/115: Basic fibroblast growth factor (bFGF, FGF-2) - C12N 2501/165: Vascular endothelial growth factor [VEGF] - C12N 2506/08: from cells of the nervous system - C12N 2506/13: from connective tissue cells, from mesenchymal cells - C12N 2506/1384: from adipose-derived stem cells [ADSC], from adipose stromal stem cells - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - 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) ## Abstract Methods of producing stem cell conditioned media to treat mammalian injuries or insults. In at least one embodiment of a method for isolating non-endothelial adipocyte-depleted stromal cells of the present disclosure, the method comprises, comprising dissociating subcutaneous adipose tissue isolated from a mammal into a cell suspension, removing adipocytes from said cell suspension, resulting in a non-endothelial adipocyte-depleted cell suspension, and culturing the non-endothelial adipocyte-depleted cell suspension in a media containing growth factors VEGF, bFGF, EGF, and IGF, such that a mixed population of cells comprising a first population of further differentiated non-endothelial adipocyte-depleted CD34+/VE−cadherin− cells and a second population of further differentiated non-endothelial adipocyte-depleted CD34+/VE−cadherin+ cells are obtained and expanded. ## Claims ### Claim 1 A method for isolating non-endothelial adipocyte-depleted stromal cells, comprising: a) dissociating subcutaneous adipose tissue isolated from a mammal into a cell suspension; b) removing adipocytes from said cell suspension, resulting in a non-endothelial adipocyte-depleted cell suspension; and c) culturing the non-endothelial adipocyte-depleted cell suspension in a media containing growth factors vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and insulin-like growth factor (IGF), such that a mixed population of cells comprising a first population of further differentiated non-endothelial adipocyte-depleted CD34+/VE−cadherin− cells and a second population of further differentiated non-endothelial adipocyte-depleted CD34+/VE−cadherin+ cells are obtained and expanded. ### Claim 2 (depends on 1) The method of claim 1, wherein the mixed population of cells secrete vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and granulocyte-colony stimulating factor (G-CSF). ### Claim 3 (depends on 1) The method of claim 1, further comprising culturing the mixed population of cells of step c) in DMEM. ### Claim 4 (depends on 1) The method of claim 1, further comprising culturing the mixed population of cells of step c) in DMEM and 10% fetal bovine serum. ### Claim 5 (depends on 1) The method of claim 1, wherein said mixed population of cells are exposed to a receptor ligand cocktail, said ligands being selected from the group consisting of at least one of vascular endothelial growth factor (VEGF), leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), insulin-like growth factor 1 (IGF1), insulin-like growth factor 2 (IGF2), hepatocyte growth factor (HGF), cardiotrophin, myotrophin, nitric oxide synthase 1, nitric oxide synthase 2, nitric oxide synthase 3, tumor necrosis factor alpha, tumor necrosis factor beta, fibroblast growth factor, pleotrophin, endothelin, and angiopoietin. ### Claim 6 (depends on 1) The method of claim 1, further comprising exposing the non-endothelial adipocyte-depleted cell suspension to red cell lysis buffer before the culturing step. ### Claim 7 (depends on 1) The method of claim 1, wherein the non-endothelial adipocyte-depleted stromal cells do not secrete detectable levels of basic fibroblast growth factor (bFGF). ### Claim 8 (depends on 1) The method of claim 1, further comprising the step of culturing the mixed population of cells in a growth factor-free basal medium without growth factors added or introduced by the manufacturer or user. ### Claim 9 (depends on 8) The method of claim 8, wherein the growth-factor free basal medium is EBM 2. ### Claim 10 (depends on 1) The method of claim 1, wherein the culturing step is performed on matrigel. ### Claim 11 (depends on 1) The method of claim 1, wherein the media further comprises 5-azacytidine. ### Claim 12 (depends on 1) The method of claim 1, wherein the dissociating step is performed using agitation, sonic energy, or thermal energy. ### Claim 13 (depends on 1) The method of claim 1, wherein the dissociating step is performed using collagenase, dispase, or trypsin. ### Claim 14 (depends on 1) The method of claim 1, further comprising introduction of an exogenous nucleic acid molecule encoding a protein of interest into the mixed population of cells of step c). ### Claim 15 A method for isolating non-endothelial adipocyte-depleted stromal cells, comprising: a) dissociating subcutaneous adipose tissue isolated from a mammal into a cell suspension; b) removing adipocytes from said cell suspension, resulting in a non-endothelial adipocyte-depleted cell suspension; and c) culturing the non-endothelial adipocyte-depleted cell suspension in a media containing growth factors vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and insulin-like growth factor (IGF), such that the cells in the non-endothelial adipocyte-depleted cell suspension are expanded. ### Claim 16 (depends on 15) The method of claim 15 wherein the concentrations of VEGF, bFGF, EGF, and IGF are no greater than that present in EGM2MV. ### Claim 17 (depends on 15) The method of claim 15 wherein the cells in the non-endothelial adipocyte-depleted cell suspension expands to at least 15000 cells per cm2 in eight days. ### Claim 18 A method for isolating non-endothelial adipocyte-depleted stromal cells, comprising: a) dissociating subcutaneous adipose tissue isolated from a mammal into a cell suspension; b) removing adipocytes from said cell suspension, resulting in a non-endothelial adipocyte-depleted cell suspension; and c) culturing the non-endothelial adipocyte-depleted cell suspension in a media containing growth factors vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and insulin-like growth factor (IGF), such that the cells are expanded and a population of pluripotent cells are obtained; d) passaging at least some of the population of pluripotent cells; and e) further expanding the passaged population of pluripotent cells. ### Claim 19 (depends on 17) The method of claim 17 wherein step e) is performed in the media of step c). ### Claim 20 (depends on 17) The method of claim 17 wherein step e) is performed in EGM2MV.