| CPC A61K 9/5031 (2013.01) [A61K 9/5123 (2013.01); A61L 27/3608 (2013.01); A61L 27/3804 (2013.01); C12N 5/0663 (2013.01); A61L 2430/38 (2013.01); C07C 31/225 (2013.01); C07C 317/04 (2013.01); C07H 3/04 (2013.01); C09D 177/04 (2013.01)] | 43 Claims |
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1. A method of making a coated biological composition comprising the steps of:
(a) grinding a cut vertebral body to produce a mixture comprising crushed vertebral bone and bone marrow, wherein the cut vertebral body is obtained from a cadaver donor;
(b) mechanically separating cellular and non-cellular components of the bone marrow from the vertebral bone in the mixture by tumbling and sieving the vertebral bone from a decanted fluid of the bone marrow comprising the cellular components comprising whole cells, and non-whole cellular components comprising non-whole cell components;
(c) concentrating the decanted fluid by centrifugation;
(d) filtering the decanted fluid through a blood filter to form a mixture including the cellular and non-whole cellular components and remove any remaining vertebral bone;
(e) separating the cellular components comprising whole cells and non-whole cellular components of the bone marrow directly from the mixture of step (d) by density gradient centrifugation, wherein the separation under centrifugation establishes a cell density gradient;
(e) collecting non-cellular fractions or non-cellular components or combinations-thereof of predetermined density from the gradient;
(f) washing the non-cellular fractions or non-cellular components or combinations thereof to create a mixture;
(g) suspending the mixture of step (f) to a predetermined concentration in a liquid protectant to form the coated biological composition;
(h) freezing the coated biological composition at a predetermined controlled rate; and
(i) thawing and freezing the coated biological composition a second time prior to storage;
(ii) thawing and refrigerating the coated biological composition;
(iii) thawing and concentrating the coated biological composition by drying; or
(iv) lyophilizing the frozen coated biological composition for ambient or room temperature storage.
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