# US 11,077,148 B2 · Acellular biologic composition and method of manufacture > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,077,148 B2 - Document ID: US11077148B2 - Status: Granted - Kind: B2 - Application number: 15/950,365 - Filed: 2018-04-11 - Published: 2018-08-16 - Granted: 2021-08-03 - Earliest claimed: 2015-07-20 - Assignees: Vivex Biologics Group, Inc. - Applicants: Vivex Biologics Group, Inc. - Inventors: Timothy Ganey; Wendy W. Weston; Gaëtan Jean-Robert Delcroix - Web page: https://patentgrep.com/patent/US11077148B2 ## CPC - A61K 35/50: Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic stem cells - C12N 5/00: Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor; (plant reproduction by tissue culture techniques A01H4/00) - C12N 5/0605: {Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly} - 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) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract A biological composition has a mixture of mechanically selected allogeneic biologic material derived from placental tissue. The mixture has non-whole cellular components including vesicular components and active and inactive components of biological activity, cell fragments, cellular excretions, cellular derivatives, and extracellular components. The mixture including non-whole cell fractions including one or more of exosomes, transcriptosomes, proteasomes, membrane rafts, lipid rafts. The mixture is compatible with biologic function. ## Claims ### Claim 1 A method of making a biological composition comprises the steps of: collecting, recovering and processing placental tissue from a donor; mechanically separating cellular and non-cellular components or combinations thereof of placental tissue; concentrating by centrifugation and filtering to form a suspension of the cellular and non-cellular components; separating by density gradient centrifugation to form a cell pellet of the cellular components and a supernatant of the non-cellular components wherein the cellular components in the cell pellet are separated from the supernatant and removed and wherein the supernatant is further consisting of filtering through a 0.45 micron filter; collecting non-cellular fractions or non-cellular components or combinations thereof of predetermined density by increasing centrifugation strength; washing the non-cellular fractions or non-cellular components or combinations thereof; quantifying the non-cellular fractions or non-cellular components or combinations thereof separated by density gradient concentration is greater than zero; suspending the non-cellular fractions or non-cellular components or combinations thereof to a concentration with a blend of collected, recovered and processed placental tissue and bone particles to create a mixture in a cryoprotectant consisting of a polyampholyte cryoprotectant to create a biological composition, wherein the bone particles having a size range of 100 to 300 μm of demineralized cortical bone, mineralized cortical bone and mineralized cancellous bone intermixed with the non-cellular fractions or non-cellular components; and freezing the biological composition at a controlled rate. ### Claim 2 (depends on 1) The method of preparing the biological composition for use made according to the method of claim 1 by the steps of: thawing the biological composition; diluting the thawed biological composition in saline without spinning; and implanting the diluted biological composition with the blend of additional tissue being intermixed by packing, injection or any other suitable means into a patient. ### Claim 3 (depends on 2) The method of preparing the biological composition for use made according to the method of claim 2 wherein the step of thawing the biological composition occurs at a temperature of 37 degrees C. for 2 to 3 minutes in a warm water bath. ### Claim 4 (depends on 1) The method of making the biological composition of claim 1 further consists of the step of adding a quantity of whole cells to the non-cellular fractions or non-cellular components or combinations to a concentration with the blend of collected, recovered and processed placental tissue and bone particles in the polyampholyte cryoprotectant to create the biological composition. ### Claim 5 A method of making a biological composition comprises the steps of: mechanically separating cellular and non-cellular components or combinations thereof of placental tissue; concentrating by centrifugation and filtering to form a suspension of the cellular and non-cellular components; separating by density gradient centrifugation to form a cell pellet of the cellular components and a supernatant of the non-cellular components wherein the cellular components in the cell pellet are separated from the supernatant and removed and wherein the supernatant is further consisting of filtering through a 0.45 micron filter; collecting non-cellular fractions or non-cellular components or combinations thereof of predetermined density by increasing centrifugation strength; washing the non-cellular fractions or non-cellular components or combinations thereof; quantifying the non-cellular fractions or non-cellular components or combinations thereof separated by density gradient concentration is greater than zero; suspending the non-cellular fractions or non-cellular components or combinations thereof to a concentration with a blend of collected and processed placental tissue to create a mixture in a cryoprotectant consisting of a polyampholyte cryoprotectant to form a biological composition; and freezing the biological composition at a controlled rate. ### Claim 6 (depends on 5) The method of preparing the biological composition for use made according to the method of claim 5 by the steps of: thawing the biological composition; diluting the thawed biological composition in saline without spinning; and implanting the diluted biological composition by packing, injection or any other suitable means into a patient. ### Claim 7 (depends on 6) The method of preparing the biological composition for use made according to the method of claim 6 wherein the step of thawing the biological composition occurs at a temperature of 37 degrees C. for 2 to 3 minutes in a warm water bath. ### Claim 8 (depends on 5) The method of making the biological composition of claim 5 further consists of the step of adding a quantity of whole cells to the non-cellular fractions or non-cellular components or combinations to a concentration with the blend of collected, recovered and processed placental tissue in the polyampholyte cryoprotectant to create the biological composition. ### Claim 9 A method of making a biological composition comprises the steps of: collecting, recovering and processing placental tissue from a donor; mechanically separating cellular and non-cellular components or combinations thereof of placental tissue; concentrating by centrifugation and filtering to form a suspension of the cellular and non-cellular components; separating by density gradient centrifugation to form a cell pellet of the cellular components and a supernatant of the non-cellular components wherein the cellular components in the cell pellet are separated from the supernatant and removed and wherein the supernatant is further consisting of filtering through a 0.45 micron filter; collecting non-cellular fractions or non-cellular components or combinations thereof of predetermined density by increasing centrifugation strength; washing the non-cellular fractions or non-cellular components or combinations thereof; quantifying the non-cellular fractions or non-cellular components or combinations thereof separated by density gradient concentration is greater than zero; suspending the non-cellular fractions or non-cellular components or combinations thereof to a concentration with a blend of collected, recovered and processed placental tissue and bone particles to create a mixture in a cryoprotectant consisting of a polyampholyte cryoprotectant to create a biological composition, wherein the bone particles having a size range of 100 to 300 μm of demineralized cortical bone, mineralized cortical bone and mineralized cancellous bone intermixed with the non-cellular fractions or non-cellular components; and freeze-drying the biological composition. ### Claim 10 (depends on 9) The method of preparing the biological composition for use of claim 9 further consists of the steps of: diluting the biological composition in saline without spinning; and implanting the diluted biological composition with the blend of additional tissue being intermixed by packing, injection or any other suitable means into a patient. ### Claim 11 (depends on 9) The method of making the biological composition of claim 9 further consists of the step of adding a quantity of whole cells to the non-cellular fractions or non-cellular components or combinations to a concentration with the blend of collected, recovered and processed placental tissue and bone particles in the polyampholyte cryoprotectant to create the biological composition. ### Claim 12 A method of making a biological composition comprises the steps of: mechanically separating cellular and non-cellular components or combinations thereof of placental tissue; concentrating by centrifugation and filtering to form a suspension of the cellular and non-cellular components; separating by density gradient centrifugation to form a cell pellet of the cellular components and a supernatant of the non-cellular components wherein the cellular components in the cell pellet are separated from the supernatant and removed and wherein the supernatant is further consisting of filtering through a 0.45 micron filter; collecting non-cellular fractions or non-cellular components or combinations thereof of predetermined density by increasing centrifugation strength; washing the non-cellular fractions or non-cellular components or combinations thereof; quantifying the non-cellular fractions or non-cellular components or combinations thereof separated by density gradient concentration is greater than zero; suspending the non-cellular fractions or non-cellular components or combinations thereof to a concentration with a blend of collected and processed placental tissue to create a mixture in a cryoprotectant consisting of a polyampholyte cryoprotectant to form a biological composition; and freeze-drying the biological composition. ### Claim 13 (depends on 12) The method of preparing the biological composition for use of claim 12 further consists of the steps of: diluting the biological composition in saline without spinning; and implanting the diluted biological composition with the blend of additional tissue being intermixed by packing, injection or any other suitable means into a patient. ### Claim 14 (depends on 12) The method of making the biological composition of claim 12 further consists of the step of adding a quantity of whole cells to the non-cellular fractions or non-cellular components or combinations to a concentration with the blend of collected, recovered and processed placental tissue in the polyampholyte cryoprotectant to create the biological composition.