Methods for making cytokine compositions from tissues using non-centrifugal methods

• US 10,208,095 B2
• Filed: 03/15/2013
• Issued: 02/19/2019
• Est. Priority Date: 03/15/2013
• Status: Active Grant

• Current Assignee: Biomet Manufacturing, LLC (Zimmer Biomet Holdings, Inc.)
• Original Assignee: Biomet Manufacturing, LLC (Zimmer Biomet Holdings, Inc.)
• Inventors: Leach, Michael D., Woodell-May, Jennifer E., Higgins, Joel C., O'Shaughnessey, Krista
• Primary Examiner(s): Ford, Vanessa L.
• Assistant Examiner(s): Dillahunt, Sandra E
• Application Number:
  US13/839,280
• Publication Number:
  US 20140274894A1
• Time in Patent Office: 2,167 Days
• Field of Search: None
• CPC Class Codes: A61K 2300/00 Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• CPC Class Codes: A61K 35/14 Blood; Artificial blood perfluorocarbons A61K31/02; umbilical cord blood A61K35/51; haemoglobin A61K38/42
• 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 35/35 Fat tissue; Adipocytes; Stromal cells; Connective tissues adipose-derived stem cells A61K35/28; collagen A61K38/39
• CPC Class Codes: A61K 38/1793 for cytokines; for lymphokines; for interferons
• CPC Class Codes: A61K 38/1808 Epidermal growth factor [EGF] urogastrone
• CPC Class Codes: A61K 38/1841 Transforming growth factor [TGF]
• CPC Class Codes: A61K 38/1858 Platelet-derived growth factor [PDGF]
• CPC Class Codes: A61K 38/1866 Vascular endothelial growth factor [VEGF]
• CPC Class Codes: A61K 38/191 Tumor necrosis factors [TNF], e.g. lymphotoxin [LT], i.e. TNF-beta
• CPC Class Codes: A61K 38/2006 IL-1
• CPC Class Codes: A61M 1/0281 Apparatus for treatment of blood or blood constituents prior to transfusion, e.g. washing, filtering or thawing
• CPC Class Codes: A61M 1/029 Separating blood components present in distinct layers in a container, not otherwise provided for containers for storing blood or blood components A61J1/05; sampling or analysing blood by separating blood components G01N33/491
• CPC Class Codes: C07K 1/34 by filtration, ultrafiltration or reverse osmosis
• CPC Class Codes: C07K 14/435 from animals; from humans
• CPC Class Codes: C07K 14/52 Cytokines; Lymphokines; Interferons
• CPC Class Codes: C07K 14/545 IL-1
• CPC Class Codes: G01N 33/5002 Partitioning blood components
• CPC Class Codes: G01N 33/5005 involving human or animal cells immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025

Abstract

Non-centrifugal methods for generating a solution rich in interleukin-1 receptor antagonist from a tissue comprising cytokine-producing cells. The solution rich in IL-1ra can also include at least one of sTNF-RI, sTNF-RII, IGF-I, EGF, HGF, PDGF-AB, PDGF-BB, VEGF, TGF-β1, and sIL-1 RII.

Claims

1. A method for generating a solution rich in interleukin-1 receptor antagonist (IL-1ra) comprising:

obtaining a liquid volume comprising white blood cells by separating white blood cells from a tissue comprising any one of;

(a) whole blood, bone marrow aspirate, adipose tissue;

(b) a fraction thereof;

or (c) a mixture of any of the foregoing, using dielectrophoretic field-flow-fractionation (DEP-FFF), wherein separating the white blood cells concentrates the white blood cells to at least about 15,000/μ

l; and

contacting the liquid volume comprising white blood cells with a solid extraction material to obtain a solution rich in IL-1ra.

2. The method according to claim 1, wherein the solution rich in IL-1ra further comprises one or more proteins selected from the group consisting of epidermal growth factor (EGF), hepatocyte growth factor (HGF), platelet-derived growth factor BB (PDGF-BB), and soluble interleukin one receptor II (sIL-1RII).

3. The method according to claim 1, wherein the solution rich in IL-1ra comprises:

(i) interleukin-1 receptor antagonist (IL-1ra) at a concentration of at least about 10,000 pg/ml;

(ii) soluble Tumor Necrosis Factor Receptor 1 (sTNF-R1) at a concentration of at least about 1,200 pg/ml; and

(iii) EGF, HGF, PDGF-BB, or soluble interleukin one receptor II (sIL-1RII) at a concentration of at least about 2,000 pg/ml.

4. The method according to claim 1, wherein the solid extraction material comprises any one of corundum, quartz, titanium, dextran, agarose, polyacrylamide, polystyrene, polyethylene, polyvinyl chloride, polypropylene, or a combination of any of the foregoing.

5. The method according to claim 4, wherein the solid extraction material comprises polyacrylamide.

6. The method according to claim 1, wherein the solid extraction material comprises a form selected from the group consisting of a bead, fiber, powder, porous material, and combinations thereof.

7. The method according to claim 1, wherein the surface of the solid extraction material is etched to increase its surface area.

8. The method according to claim 1, wherein the contacting is for a period of time from about 30 seconds to about 2 hours.

9. The method according to claim 1, wherein the contacting comprises subjecting the liquid to an electromagnetic field.

10. The method according to claim 9, wherein the electromagnetic field comprises a pulsed electromagnetic field or a capacitively coupled electromagnetic field.

11. A method of preparing an enriched biological solution, comprising:

(a) separating white blood cells from a tissue selected from whole blood, bone marrow aspirate, adipose tissue, a fraction thereof, or a mixture thereof to produce a volume of concentrated white blood cells, including subjecting the tissue to di electrophoretic field-flow-fractionation (DEP-FFF), wherein the separating separates at least about one-half of red blood cells in the tissue from the volume of white blood cells;

(b) mixing the volume of white blood cells with a liquid medium to form a cytokine cell suspension, wherein the cytokine cell suspension comprises white blood cells, and at least one of platelets, adipose stromal cells, and mesenchymal stromal cells;

(c) contacting the cytokine cell suspension with polyacrylamide beads for a period of time between about 30 seconds and about 72 hours; and

(d) separating liquid from the polyacrylamide beads to obtain a solution rich in IL-1ra and rich in at least one of epidermal growth factor (EGF), hepatocyte growth factor (HGF), platelet-derived growth factor-BB (PDGF-BB), and soluble interleukin-1 receptor II (sIL-1RII).

12. The method according to claim 11, wherein the tissue is autologous with the subject.

13. The method according to claim 11, wherein the solution rich in IL-1ra has an IL-1ra concentration of at least about 10,000 pg/ml.

14. The method according to claim 11, wherein the solution rich in IL-1ra further comprises at least one protein selected from soluble tumor necrosis factor receptor II (sTNF-RII), insulin-like growth factor-I (IGF-I), platelet-derived growth factor-AB (PDGF-AB), vascular endothelial growth factor (VEGF), and transforming growth factor-β

1 (TGF-β

1), wherein the concentration of the selected protein in the solution is greater than the concentration of the selected protein in normal blood.

15. The method according to claim 11, wherein separating comprises:

(a) diluting the tissue comprising white blood cells in a sucrose buffer to form a biological sample;

(b) loading the biological sample into a chamber comprising a top plate and a bottom plate, wherein the top plate and bottom plate are coupled to microelectrodes;

(c) applying a dielectrophoretic (DEP) field to the biological sample, wherein white blood cells are suspended in the biological sample; and

(d) isolating a volume of white blood cells by flowing a carrier fluid through the chamber.

16. The method according to claim 15, wherein the DEP field comprises a frequency of about 10 kHz.

17. The method according to claim 11, wherein the white blood cells are concentrated to at least about 15,000/μ

l.

18. The method according to claim 11, further comprising:

(e) administering the solution rich in IL-1ra to a mammalian subject.

19. A method for generating a biological solution enriched in anti-inflammatory cytokines and growth factors, the method comprising:

subjecting a tissue comprising any one of;

(a) whole blood, bone marrow aspirate, adipose tissue;

(b) a fraction thereof;

or (c) a combination of any of the foregoing, to dielectrophoretic field-flow-fractionation to separate white blood cells from the tissue to produce a volume of white blood cells at a concentration of at least about 15,000/μ

l, wherein the volume of white blood cells includes about one-half or less of red blood cells from the tissue; and

contacting the volume of white blood cells with a solid extraction material to obtain the biological solution enriched in at least one protein selected from interleukin-1 receptor II, platelet-derived growth factor BB, epidermal growth factor, and hepatocyte growth factor.

20. The method of claim 19, wherein the at least protein is at a concentration of at least 2,000 pg/ml.

21. The method of claim 19, wherein the at least one protein is present in a concentration greater than the concentration of the at least one protein in normal blood.

22. The method of claim 19, wherein the at least one protein is a growth factor at a concentration of at least about 1,500 pg/ml.

23. The method of claim 19, wherein the solution further comprises at least one supplemental protein selected from soluble tumor necrosis factor receptor II (sTNF-RII), insulin-like growth factor-I (IGF-I), platelet-derived growth factor-AB (PDGF-AB), vascular endothelial growth factor (VEGF), and transforming growth factor-β

1 (TGF-β

1), wherein the concentration of the selected supplemental protein in the solution is greater than the concentration of the selected supplemental protein in normal blood.

24. The method of claim 19, wherein the contacting comprises subjecting the white blood cells to an electromagnetic field.

25. The method of claim 19, wherein the contacting is for a period of less than 30 minutes.

26. The method according to claim 19, wherein the solid extraction material comprises any one of corundum, quartz, titanium, dextran, agarose, polyacrylamide, polystyrene, polyethylene, polyvinyl chloride, polypropylene, or a combination of any of the foregoing.