Acellular biologic composition and method of manufacture
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.
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.
2. 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.
3. 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.
4. 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.
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.
6. 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.
7. 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.
8. 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.
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.
10. 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.
11. 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.
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.
13. 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.
14. 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.