# US 7,931,938 B2 · Diamond-like carbon-coated cell culture substrates > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 7,931,938 B2 - Document ID: US7931938B2 - Status: Granted - Kind: B2 - Application number: 10/575,247 - Filed: 2004-10-08 - Published: 2007-11-29 - Granted: 2011-04-26 - Earliest claimed: 2003-10-10 - Assignees: Cellular Bioengineering, Inc. - Inventors: Ge Ming Lui - Web page: https://patentgrep.com/patent/US7931938B2 ## CPC - A61L 27/3878: {Nerve tissue, brain, spinal cord, nerves, dura mater} - A61L 27/303: {Carbon} - A61L 27/3633: {Extracellular matrix [ECM]} - A61L 27/383: {Nerve cells, e.g. dendritic cells, Schwann cells} - A61L 27/3895: {using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions} - A61P 25/00: Drugs for disorders of the nervous system - C12M 25/14: {Scaffolds; Matrices (in general C12N5/0068)} - C12N 5/0068: {General culture methods using substrates (for specific animal cell type C12N5/06)} - C12N 5/0621: {Eye cells, e.g. cornea, iris pigmented cells (photoreceptors C12N5/062)} - C12N 2502/28: Vascular endothelial cells - C12N 2533/10: Mineral substrates - C12N 2533/30: Synthetic polymers - C12N 2533/50: Proteins - C12N 2533/54: Collagen; Gelatin - 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) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - 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} - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract This present invention describes a method of coating a polymer surface with diamond-like carbon (DLC) to render it useful as a carrier for cells derived from neural crest origin, preferable neuronal cells that form dendrites. The biopolymer to be coated with the DLC will include biodegradable polymers and other implantable biopolymers to act as a carrier system for cell transplantation into the various parts of the body, including the brain, the eye, the central and peripheral nervous system, the lung, the liver, the spleen, the kidney, and the bone and cartilage. The biopolymer can be in sheet form or microparticle form, and can be imbedded with, or incorporated into during its synthesis, attachment or growth promoting reagents to enhance and support neuraonal call attachment and growth. This coating method can also augment other coating agents such as extracellular matrix (ECM) secreted by cultured bovine corneal endothelial cells, as well as adhesive molecules such as fibronectin, laminin, and RGDS. The coating step can be a sequential process where the DLC layer will be added on top of an ECM coated surface or an attachment factor coated surface. ## Claims ### Claim 1 A cell culture substrate, comprising a biodegradable biopolymer coated with hydrogen free diamond-like carbon, wherein cells can attach to and grow on the surface of the hydrogen free diamond-like carbon. ### Claim 2 (depends on 1) The cell culture substrate of claim 1, wherein the biopolymer is in a sheet form. ### Claim 3 (depends on 1) The cell culture substrate of claim 1, wherein the biopolymer is in micro particle form. ### Claim 4 (depends on 1) The cell culture substrate of claim 1, wherein the biopolymer has embedded or incorporated into it during its synthesis one or more attachment reagents selected from the group consisting of: laminin, fibronectin, RGDS (SEQ ID NO: 1), basic fibroblast growth factor (bFGF) conjugated with polycarbophyll, epidermal growth factor (EGF) conjugated with polycarbophyll, and heparin sulfate. ### Claim 5 (depends on 1) A method of growing neurons in culture comprising: providing the cell culture substrate of claim 1; seeding neurons onto the hydrogen free diamond-like carbon; and culturing the neurons. ### Claim 6 (depends on 5) The method of claim 5, wherein the biopolymer is in sheet form. ### Claim 7 (depends on 5) The method of claim 5, wherein the biopolymer is in micro particle form. ### Claim 8 (depends on 4) A method of growing neurons in culture comprising: providing the cell culture substrate of claim 4; seeding neurons onto the hydrogen free diamond-like carbon; and culturing the neurons. ### Claim 9 A three dimensional cell culture substrate comprising a semi-solid biopolymer coated with Diamond-Like Carbon, wherein the biopolymer is comprised of chitosan or sodium alginate, and wherein neural cells can attach to and grow on the surface of the Diamond-Like Carbon. ### Claim 10 (depends on 9) The cell culture substrate of claim 9, wherein the biopolymer has embedded or incorporated into it during its synthesis one or more attachment reagents selected from the group consisting of laminin, fibronectin, RGDS (SEQ ID NO: 1), basic fibroblast growth factor (bFGF) conjugated with polycarbophyll, epidermal growth factor (EGF) conjugated with polycarbophyll, heparin sulfate, and nerve growth factor (NGF), in an amount sufficient to allow neural or nerve cells transplanted onto the growth medium at low density to proliferate and send out neural processes. ### Claim 11 (depends on 10) The cell culture substrate of claim 10, wherein said biopolymer is shaped into beads, sheets or micro-particles. ### Claim 12 (depends on 10) A method of transplanting neurons to a recipient comprising: providing the three dimensional cell culture substrate of claim 10; seeding neurons of interest onto the diamond-like carbon; allowing the neurons to grow to sufficient density; and implanting the neurons on the cell culture substrate into said recipient. ### Claim 13 A three dimensional cell culture substrate comprising a semi-solid biopolymer coated with bovine corneal epithelial cell-extracellular matrix (BCE-ECM) and the BCE-ECM is further coated with Diamond-Like Carbon, wherein neural cells can attach to and grow on the surface of the Diamond-Like Carbon. ### Claim 14 (depends on 13) The cell culture substrate of claim 13, wherein the biopolymer is comprised of chitosan or sodium alginate. ### Claim 15 (depends on 13) The cell culture substrate of claim 13, wherein the biopolymer has embedded or incorporated into it during its synthesis one or more attachment reagents selected from the group consisting of: laminin, fibronectin, RGDS (SEQ ID NO: 1), basic fibroblast growth factor (bFGF) conjugated with polycarbophyll, epidermal growth factor (EGF) conjugated with polycarbophyll, heparin sulfate, and nerve growth factor (NGF), in an amount sufficient to allow neural or nerve cells transplanted onto the substrate at low density to proliferate and send out neural processes. ### Claim 16 (depends on 15) The cell culture substrate of claim 15, wherein said biopolymer is shaped into beads, sheets or micro-particles. ### Claim 17 A laboratory apparatus having a coating suitable for inducing the growth and attachment of cells, the apparatus having an inside and outside surface, wherein the inside surface is the surface in contact with cells and cellular media and the inside surface of said apparatus is coated with a film of Diamond-like-Carbon layered over a biopolymer coating. ### Claim 18 (depends on 17) The apparatus of claim 17, wherein the apparatus is selected from the group consisting of cell culture dishes, petri dishes, tissue culture flasks, plates, bottles, slides, filter chambers, slide chambers, roller bottles, harvesters and tubing. ### Claim 19 A laboratory apparatus having a coating suitable for inducing the growth and attachment of cells, the apparatus having an inside surface and an outside surface, wherein the inside surface is the surface in contact with cells and cellular media and the inside surface of said apparatus is coated with a film of Diamond-like-Carbon, the Diamond-like-Carbon being layered over a biopolymer coating and at least one other coating. ### Claim 20 (depends on 19) The apparatus of claim 19, wherein the at least one other coating is an extracellular matrix. ### Claim 21 (depends on 20) The apparatus of claim 20, wherein the coating is BCE-ECM. ### Claim 22 A method of making a laboratory apparatus suitable for inducing the growth and attachment of cells, the method comprising: a) obtaining a laboratory apparatus having an inside surface and an outside surface, wherein the inside surface is to be in contact with cells and cellular media; b) applying to the inside surface of the apparatus a biopolymer coating; then c) applying a film of Diamond-like-Carbon over the biopolymer coating. ### Claim 23 (depends on 22) The method of claim 22, further comprising between steps b) and c), step b′) applying at least one other coating over the biopolymer coating.