# US 10,246,678 B2 · Biomatrix scaffolds > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,246,678 B2 - Document ID: US10246678B2 - Status: Granted - Kind: B2 - Application number: 15/221,413 - Filed: 2016-07-27 - Published: 2017-01-19 - Granted: 2019-04-02 - Earliest claimed: 2010-07-02 - Assignees: The University of North Carolina at Chapel Hill - Applicants: The University of North Carolina at Chapel Hill - Inventors: Yunfang Wang; Lola Cynthia McAdams Reid; Mitsuo Yamauchi; Cai-Bin Cui; Andrew Zhuang Wang; Michael Edward Werner - Web page: https://patentgrep.com/patent/US10246678B2 ## CPC - C12N 5/0068: {General culture methods using substrates (for specific animal cell type C12N5/06)} - A61L 27/24: Collagen - A61L 27/3604: {characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel} - A61L 27/3683: {subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment} - A61L 27/3691: {characterised by physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation} - C12N 5/067: {Hepatocytes} - C12N 5/0671: {Three-dimensional culture, tissue culture or organ culture; Encapsulated cells} - C12N 5/0693: {Tumour cells; Cancer cells} - C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00) - A61L 2430/28: for liver reconstruction - C12N 2500/25: Insulin-transferrin; Insulin-transferrin-selenium - C12N 2500/36: Lipids - C12N 2501/20: Cytokines; Chemokines - C12N 2501/998: Proteins not provided for elsewhere - C12N 2506/14: from hepatocytes - C12N 2533/54: Collagen; Gelatin - C12N 2533/90: Substrates of biological origin, e.g. extracellular matrix, decellularised tissue - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - A61L: METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES (preservation of bodies or disinfecting characterised by the agents employed A01N; preserving, e.g. sterilising, food or foodstuffs A23; preparations for medical, dental or toiletry purposes A61K) ## Abstract The present invention provides biomatrix scaffolds, a tissue extract enriched for extracellular matrix components and bound growth factors, cytokines and hormones, and methods of making and using same. ## Claims ### Claim 1 A biomatrix scaffold produced by a method comprising: a) perfusing the biological tissue or homogenizing the biological tissue from the group consisting of liver, lung, thyroid, skin, pancreas, blood vessels, bladder, kidneys, brain, biliary tree, duodenum, abdominal aorta, iliac vein, heart, and intestines, with a first medium, wherein the osmolality of said first medium is from about 250 mOsm/kg to about 350 mOsm/kg and said first medium is serum free and at neutral pH; then b) perfusing the biological tissue or extracting the homogenate of step (a) with a delipidating buffer comprising lipases and/or detergents in said first medium, wherein the detergents do not comprise sodium dodecyl sulfate or Triton X-100; then c) perfusing the tissue or extracting the homogenate of step (b) with a buffer at a neutral pH and comprising a salt concentration from about 2.0 M to about 5.0 M NaCl; then d) rinsing the tissue or homogenate of step (c) with a second medium that is at neutral pH, is serum-free and has an osomolality from about 250 mOsm/kg to about 350 mOsm/kg, thereby producing an intact or homogenized biomatrix scaffold from the biological tissue, said biomatrix scaffold comprising at least 95% of the collagens and most collagen-associated matrix components and matrix bound growth factors, hormones and cytokines of the biological tissue. ### Claim 2 (depends on 1) A method of producing a cell culture, comprising: a) contacting the biomatrix scaffold of claim 1 with cell culture medium in a culture apparatus; and b) seeding the biomatrix scaffold of step (a) with cells, thereby producing a cell culture. ### Claim 3 (depends on 2) The method of claim 2, wherein the cell culture medium comprises at least one of the constituents present in interstitial fluid, wherein the osmolality of said medium is from about 250 mOsm/kg to about 350 mOsm/kg and wherein said medium is serum free. ### Claim 4 (depends on 3) The method of claim 3, wherein the medium is RPMI-1640. ### Claim 5 (depends on 2) The method of claim 2, wherein the cells are of the same type as the cells of the biological tissue used to make the biomatrix scaffold. ### Claim 6 (depends on 2) The method of claim 2, wherein the cells are selected from the group consisting of embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, determined stem cells, perinatal stem cells, amniotic fluid-derived stem cells (AFSCs), mesenchymal stem cells (MSCs) from any source, committed progenitors or adult cells of any tissue type, mature cells, normal cells, diseased cells, tumor cells and any combination thereof. ### Claim 7 (depends on 2) The method of claim 2, wherein the cells are selected from the group consisting of liver cells, parenchymal cells, stellate cells, endothelial cells, hepatocytes, cholangiocytes, biliary tree cells that are not cholangiocytes and pancreatic cells. ### Claim 8 (depends on 2) A method of enhancing and accelerating differentiation of stem cells and/or progenitors to mature cells, comprising producing a cell culture according to the method of claim 2, wherein the cells are stem cells and the cell culture medium is formulated for mature cells, thereby enhancing and accelerating differentiation of stem cells and/or progenitors to mature cells. ### Claim 9 (depends on 8) The method of claim 8, wherein the cells are adult cells of any type or are stem cells or progenitors selected from the group consisting of embryonic stem cells, induced pluripotent stem cells, germ layer stem cells, determined stem cells, perinatal stem cells, amniotic fluid-derived stem cells, mesenchymal stem cells, transit amplifying cells or committed progenitors of any tissue type. ### Claim 10 (depends on 8) The method of claim 8, wherein the cell culture medium is selected from the group consisting of RPMI 1640, DME/F12, DME, F12, Waymouth's, and William's medium. ### Claim 11 (depends on 1) A method of producing a cell culture, comprising: a) freezing the biomatrix scaffold of claim 1; b) preparing a frozen section from the biomatrix scaffold of step (a) as a cell culture sub stratum; c) contacting the cell culture substratum of step (b) with cell culture medium in a culture apparatus; and d) seeding the cell culture substratum of step (c) with cells, thereby producing a cell culture. ### Claim 12 (depends on 11) The method of claim 11, wherein the cell culture-medium comprises at least one of the constituents present in interstitial fluid, wherein the osmolality of said medium is from about 250 mOsm/kg to about 350 mOsm/kg and wherein said medium is serum free. ### Claim 13 (depends on 12) The method of claim 12, wherein the medium is RPMI-1640. ### Claim 14 (depends on 11) The method of method of claim 11, wherein the cells are of the same type as the cells of the biological tissue used to make the biomatrix scaffold. ### Claim 15 (depends on 11) The method of claim 11, wherein the cells are selected from the group consisting of embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, determined stem cells, perinatal stem cells, amniotic fluid-derived stem cells (AFSCs), mesenchymal stem cells (MSCs) from any source, committed progenitors or adult cells of any tissue type, mature cells, normal cells, diseased cells, tumor cells and any combination thereof. ### Claim 16 (depends on 11) The method of claim 11, wherein the cells are selected from the group consisting of liver cells, parenchymal cells, stellate cells, endothelial cells, hepatocytes, cholangiocytes, biliary tree cells that are not cholangiocytes and pancreatic cells. ### Claim 17 (depends on 11) A method of enhancing and accelerating differentiation of stem cells and/or progenitors to mature cells, comprising producing a cell culture according to the method of claim 11, wherein the cells are stem cells and the cell culture medium is formulated for mature cells, thereby enhancing and accelerating differentiation of stem cells and/or progenitors to mature cells. ### Claim 18 (depends on 17) The method of claim 17, wherein the cells are adult cells of any type or are stem cells or progenitors selected from the group consisting of embryonic stem cells, induced pluripotent stem cells, germ layer stem cells, determined stem cells, perinatal stem cells, amniotic fluid-derived stem cells, mesenchymal stem cells, transit amplifying cells or committed progenitors of any tissue type. ### Claim 19 (depends on 17) The method of claim 17, wherein the cell culture medium is selected from the group consisting of RPMI 1640, DME/F12, DME, F12, Waymouth's, and William's medium. ### Claim 20 (depends on 1) A method of producing a cell culture, comprising: a) grinding the biomatrix scaffold of claim 1 to a powder; b) coating at least part of a culture apparatus with the powder of step (a) to produce a cell culture substratum; c) contacting the cell culture substratum of (b) with cell culture medium in the culture apparatus; and d) seeding the cell culture substratum of (c) with cells, thereby producing a cell culture. ### Claim 21 (depends on 20) The method of claim 20, wherein the grinding of the biomatrix scaffold is carried out in a freezer mill at or near liquid nitrogen temperatures. ### Claim 22 (depends on 1) A method of producing virus particles of a lineage dependent virus, comprising: a) contacting the biomatrix scaffold of claim 1 with cell culture medium in a culture apparatus; b) seeding the biomatrix scaffold of (a) with cells of a type and lineage stage that can be infected with the lineage dependent virus; c) infecting the cells of (b) with the lineage dependent virus; d) maintaining the infected cells on the biomatrix scaffold under culture conditions; and e) collecting virus particles produced in the infected cells, thereby producing virus particles of the lineage dependent virus. ### Claim 23 (depends on 22) The method of claim 22, wherein the lineage dependent virus is selected from the group consisting of hepatitis C virus, hepatitis B virus, norovirus, and human papilloma virus. ### Claim 24 (depends on 1) A method of producing an organoid formed by recellularization of a biomatrix scaffold, comprising: a) contacting the biomatrix scaffold of claim 1 with cell culture medium in a culture apparatus; b) seeding the biomatrix scaffold of (a) with cells of the same tissue type as the biological tissue used to prepare the biomatrix scaffold; and c) maintaining the cells on the biomatrix scaffold under culture conditions, whereby organoids form from the cells, thereby producing an organoid formed by recellularization of the biomatrix scaffold. ### Claim 25 (depends on 24) The method of claim 24, further comprising the step of contacting the organoid with a subject, for use as an assist device. ### Claim 26 (depends on 24) The method of claim 24, wherein the cells are liver cells. ### Claim 27 A cell culture substratum produced by a method comprising: a) perfusing the biological tissue or homogenizing the biological tissue with a first medium, wherein the osmolality of said first medium is from about 250 mOsm/kg to about 350 mOsm/kg and said first medium is serum free and at neutral pH; then b) perfusing the biological tissue or extracting the homogenate of step (a) with a delipidating buffer comprising lipases and/or detergents in said first medium, wherein the detergents do not comprise sodium dodecyl sulfate or Triton X-100; then c) perfusing the tissue or extracting the homogenate of step (b) with a buffer at a neutral pH and comprising a salt concentration from about 2.0 M to about 5.0 M NaCl; then d) rinsing the tissue or homogenate of step (c) with a second medium that is at neutral pH, is serum-free and has an osomolality from about 250 mOsm/kg to about 350 mOsm/kg, thereby producing an intact or homogenized biomatrix scaffold from the biological tissue, said biomatrix scaffold comprising at least 95% of the collagens and most collagen-associated matrix components and matrix bound growth factors, hormones and cytokines of the biological tissue e) freezing the biomatrix scaffold; f) preparing a frozen section from the biomatrix scaffold of step (e) as a cell culture substratum.