Compositions for biological systems and methods for preparing and using the same

• US 10,363,271 B2
• Filed: 05/24/2018
• Issued: 07/30/2019
• Est. Priority Date: 06/13/2016
• Status: Expired due to Fees

• Current Assignee: Smart Surgical, Inc.
• Original Assignee: Smart Surgical, Inc.
• Inventors: Jones, Christopher D., Quintanar, Nathan
• Primary Examiner(s): Hobbs, Lisa J
• Application Number:
  US15/988,855
• Publication Number:
  US 20180264041A1
• Time in Patent Office: 432 Days
• CPC Class Codes: A61K 2035/124 the cells being hematopoietic, bone marrow derived or blood cells
• CPC Class Codes: A61K 35/15 Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells presenting a specific antigen A61K39/00; therapeutic combinations of antibodies, or fragments thereof, and blood-derived cells A61K39/00
• CPC Class Codes: A61K 35/17 Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes when activated by a specific antigen A61K39/00
• CPC Class Codes: A61K 35/28 Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
• CPC Class Codes: A61K 35/51 Umbilical cord; Umbilical cord blood; Umbilical stem cells
• CPC Class Codes: A61K 38/18 Growth factors; Growth regulators
• CPC Class Codes: A61K 47/02 Inorganic compounds
• CPC Class Codes: A61K 47/20 containing sulfur, e.g. dimethyl sulfoxide [DMSO], docusate, sodium lauryl sulfate or aminosulfonic acids
• CPC Class Codes: A61K 47/26 Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
• CPC Class Codes: A61K 47/42 Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein oligopeptides having up to five amino acids A61K47/183; polyamino acids A61K47/34
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: C12N 2500/34 Sugars
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 5/0037 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 5/0665 Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood

Abstract

A method of making a composition for influencing biological growth includes obtaining a sample comprising human umbilical cord blood and having a first volume V_UC, combining the sample with a quantity of PrepaCyte-CB or the equivalent having a volume V_PC, wherein the ratio V_UC/V_PC is between about 0.60 and about 1.75 such that a supernatant is formed, layering within a container at least a portion of the supernatant over a layer of a reagent configured for isolating mononuclear cells, the at least a portion of the supernatant having a volume V_S and the layer of the reagent having a volume V_R, wherein the volume of the layer of the reagent V_R is at least 67% the volume V_S of the at least a portion of the supernatant, placing a sufficient centrifugal force on the contents of the container to cause at least a new layer substantially comprising mononuclear cells to form, combining at least some of the layer substantially comprising mononuclear cells with lactated Ringer'"'"'s solution or the equivalent to create a mononuclear cell solution, determining the total number of live cells N_L in at least a portion of the mononuclear cell solution, the at least a portion of the mononuclear solution having a volume V_M, and adding a volume V_D of Low Molecular Weight Dextran in Dextrose solution to the at least a portion of the mononuclear cell solution, the volume V_D based at least in part on the total number of live cells N_L.

Claims

1. A method of making a composition for influencing biological growth configured for implantation within a subject, the method comprising:

(A) obtaining a sample comprising human umbilical cord blood and having a first volume V_UC, wherein the human umbilical cord blood includes red blood cells, cells other than red blood cells, and non-cellular material;

(B) combining the sample comprising human umbilical cord blood with a quantity of an aggregation agent or sedimentation agent having a volume V_PC, wherein the ratio V_UC/V_PC is between about 0.60 and about 1.75 such that a supernatant is formed;

(C) layering within a container at least a portion of the supernatant over a layer of an isolation reagent, the at least a portion of the supernatant having a volume V_S and the layer of the isolation reagent having a volume V_R, wherein the volume of the layer of the isolation reagent V_R is at least 67% the volume V_S of the at least a portion of the supernatant;

(D) placing at least about 350 times the acceleration due to gravity on the contents of the container with a force vector directed toward a closed end of the container to safely cause at least a new layer substantially comprising mononuclear cells to form;

(E) combining at least some of the layer substantially comprising mononuclear cells with lactated Ringer'"'"'s solution or an equivalent balanced or buffered solution to create a mononuclear cell solution;

(F) determining the total number of live cells N_L in at least a portion of the mononuclear cell solution, the at least a portion of the mononuclear solution having a volume V_M; and

(G) adding a volume V_DD of Low Molecular Weight Dextran in Dextrose solution to the at least a portion of the mononuclear cell solution, the volume V_DD based at least in part on the total number N_L of live cells and the anticipated future viability of the live cells.

2. The method of claim 1, further comprising, adding albumin after step (E).

3. The method of claim 1, wherein following step (G), the composition has a concentration of between 100,000 and 75 million live mononuclear cells per ml.

4. The method of claim 1, further comprising:

adjusting the pH after step (G) so that it is between 4.1 and 8.5.

5. The method of claim 1, wherein the volume of the layer of the isolation reagent V_R in step (C) is at least 75% the volume V_S of the at least a portion of the supernatant.

6. The method of claim 5, wherein the volume of the layer of the isolation reagent V_R in step (C) is at least 85% the volume V_S of the at least a portion of the supernatant.

7. The method of claim 1, wherein the ratio V_UC/V_PC in step (B) is between about 0.75 and about 1.25.

8. The method of claim 1, wherein the ratio V_UC/V_PC in step (B) is between about 1.25 and about 1.75.

9. The method of claim 1, wherein the at least a portion of the mononuclear cell solution comprises stem cells.

10. The method of claim 1, further comprising cryogenically storing the composition after step (G), wherein the cell count is reduced to between about 2.5 million live mononuclear cells and about 10.2 million live mononuclear cells per ml.

11. The method of claim 10, wherein the cell count is reduced to between about 9.8 million live mononuclear cells and about 10.2 million live mononuclear cells per ml.

12. The method of claim 1, wherein the Low Molecular Weight Dextran in Dextrose solution comprises LMD (Dextran 40).

13. The method of claim 1, wherein the composition comprises one or more bioactive agents.

14. The method of claim 13, wherein the one or more bioactive agents comprise one or more cytokines.

15. The method of claim 13, wherein the one or more bioactive agents comprise one or more growth factors.

16. The method of claim 13, wherein the one or more bioactive agents are from the sample comprising human umbilical cord blood.

17. The method of claim 13, wherein the one or more bioactive agents are from human umbilical cord blood that is not from the sample comprising human umbilical cord blood.

18. The method of claim 13, wherein the one or more bioactive agents are from commercially available human albumin or commercially available human albumin serum.

37. The method of claim 2, wherein the albumin is added to create a concentration of between about 10 mg/ml and about 150 mg/ml.

19. A method of making a composition for influencing biological growth configured for implantation within a subject, the method comprising:

(A) obtaining a sample comprising human umbilical cord blood and having a first volume V_UC, wherein the human umbilical cord blood includes red blood cells, cells other than red blood cells, and non-cellular material;

(B) combining the sample comprising human umbilical cord blood with a quantity of an aggregation agent or sedimentation agent having a volume V_PC, wherein the ratio V_UC/V_PC is between about 0.60 and about 1.75 such that a supernatant is formed;

(C) layering within a container at least a portion of the supernatant over a layer of an isolation reagent, the at least a portion of the supernatant having a layer height CHs and the layer of the isolation reagent having a layer height CH_R, wherein the layer height CH_R of the layer of the isolation reagent is at least 67% the layer height CH_S of the at least a portion of the supernatant;

(D) placing at least about 350 times the acceleration due to gravity on the contents of the container with a force vector directed toward a closed end of the container to safely cause at least a new layer substantially comprising mononuclear cells to form;

(E) combining at least some of the layer substantially comprising mononuclear cells with lactated Ringer'"'"'s solution or an equivalent balanced or buffered solution to create a mononuclear cell solution;

(F) determining the total number of live cells N_L in at least a portion of the mononuclear cell solution, the at least a portion of the mononuclear solution having a volume V_M; and

(G) adding a volume V_DD of Low Molecular Weight Dextran in Dextrose solution to the at least a portion of the mononuclear cell solution, the volume V_DD based at least in part on the total number N_L of live cells and the anticipated future viability of the live cells.

20. The method of claim 19, further comprising, adding albumin after step (E).

21. The method of claim 19, wherein following step (G), the composition has a concentration of between 100,000 and 75 million live mononuclear cells per ml.

22. The method of claim 19, further comprising:

adjusting the pH after step (G) so that it is between 4.1 and 8.5.

23. The method of claim 19, wherein the layer height CH_R of the layer of the isolation reagent in step (C) is at least 75% the layer height CH_S of the at least a portion of the supernatant.

24. The method of claim 23, wherein the layer height CH_R of the layer of the isolation reagent in step (C) is at least 85% the layer height CH_S of the at least a portion of the supernatant.

25. The method of claim 19, wherein the ratio V_UC/V_PC in step (B) is between about 0.75 and about 1.25.

26. The method of claim 19, wherein the ratio V_UC/V_PC in step (B) is between about 1.25 and about 1.75.

27. The method of claim 19, wherein the at least a portion of the mononuclear cell solution comprises stem cells.

28. The method of claim 19, further comprising cryogenically storing the composition after step (G), wherein the cell count is reduced to between about 2.5 million live mononuclear cells and about 10.2 million live mononuclear cells per ml.

29. The method of claim 28, wherein the cell count is reduced to between about 9.8 million live mononuclear cells and about 10.2 million live mononuclear cells per ml.

30. The method of claim 19, wherein the Low Molecular Weight Dextran in Dextrose solution comprises LMD (Dextran 40).

31. The method of claim 19, wherein the composition comprises one or more bioactive agents.

32. The method of claim 31, wherein the one or more bioactive agents comprise one or more cytokines.

33. The method of claim 31, wherein the one or more bioactive agents comprise one or more growth factors.

34. The method of claim 31, wherein the one or more bioactive agents are from the sample comprising human umbilical cord blood.

35. The method of claim 31, wherein the one or more bioactive agents are from human umbilical cord blood that is not from the sample comprising human umbilical cord blood.

36. The method of claim 31, wherein the one or more bioactive agents are from commercially available human albumin or commercially available human albumin serum.

38. The method of claim 20, wherein the albumin is added to create a concentration of between about 10 mg/ml and about 150 mg/ml.