Cell purification and delivery using shear thinning gel

Abstract

The invention provides a cell binding composition comprising a shear thinning gel wherein the shear thinning gel having attached to it one or more cell selective binding agents, or the shear thinning gel having dispersed therein a plurality of gel beads, the gel beads having attached to them one or more cell selective binding agents. Methods of enriching cells using the compositions and using the cells to treat injury or disease are also provided.

Claims

1. A cell binding composition comprising a shear thinning gel, wherein the shear thinning gel comprises a plurality of gel beads dispersed within the shear thinning gel, the gel beads having attached to them one or more cell selective binding agent, wherein the shear thinning gel and the gel beads are independently selected from the group consisting of alginate, gellan gum, gelatin, collagen, poly(alkylene oxide), poly(ethylene oxide), poly(ethylene-co-propylene oxide), or carboxymethyl cellulose.

2. A composition according to claim 1, wherein the cell selective binding agent is an antibody or receptor or a fragment thereof, capable of binding a predetermined antigen or ligand on a cell.

3. A composition according to claim 1, wherein the cell selective binding agent is capable of binding an osteocyte, chondrocyte or blood platelet.

4. A composition according to claim 1, wherein the cell selective binding agent is an antibody or fragment thereof which is capable of specifically binding CD34, CD29, CD44, CD73, CD105 or CD271.

5. A composition according to claim 1, additionally comprising calcium pyrophosphate particles dispersed within the composition.

6. A composition according to claim 5, wherein the calcium pyrophosphate particles are star shaped.

7. A composition according to claim 1, comprising one or more cells bound to the gel beads.

8. A delivery device comprising a composition according to claim 7.

9. The delivery device according to claim 8, wherein the delivery device is any one of a pipette, syringe, needle, spray delivery device, ointment, or cream.

10. The composition according to claim 7, wherein the one or more cells are allogenic, autologous, or xenogeneic.

11. A purification column comprising a composition according to claim 1.

12. A method of enriching a population of cells comprising providing a composition according to claim 1, contacting a sample containing the cells with the composition, allowing the cells to bind to the composition, and removing unbound samples from the composition.

13. A method according to claim 12, wherein cells bound to the composition are placed in a delivery device for delivery to a site to be treated in a patient.

14. A method according to claim 13, wherein the delivery device comprises a pipette, a syringe, a needle, a spray delivery device, an ointment or a cream.

15. A method according to claim 12, wherein the sample comprises blood, plasma, serum or bone marrow extract.

16. A method according to claim 12, wherein the cell is an osteocyte, chondrocyte or platelet.

17. A method according to claim 12, wherein the enriched cells are contacted with a site on a patient to be treated.

18. A method of treating a patient comprising administering to a patient in need thereof a therapeutically effective amount of a composition according to claim 1.

19. A method according to claim 18, wherein the method is a method of treating tendon, bone, cornea, cartilage or wound.

20. A bone repair composition comprising a shear thinning gel, wherein the shear thinning gel comprises a plurality of gel beads dispersed within the shear thinning gel, the gel beads having attached to them one or more cell selective binding agents and calcium pyrophosphate particles dispersed within the composition, and optionally one or more antibiotics.

21. A composition according to claim 20 comprising amorphous or star-shaped calcium pyrophosphate particles.

22. A composition according to claim 20, wherein the gel beads are selected from the group consisting of alginate, gellan gum, gelatin, and collagen.

Description

(1) The invention will now be described by way of example only with reference to the following figures:

(2) FIG. 1: Visual demonstration of the shear-thinning properties of an example formulation, making reference to the viscosity-shear rate profile (C). At rest, the system has shape maintaining properties even under inversion (A). Upon application of shear force, by shaking in this example, the system reduces in viscosity and gains the ability to flow out of its container (B). The viscosity profile of toothpaste is displayed to put the measured viscosity data of the example formulation in to everyday context

(3) FIG. 2: Photo showing the example gel formulation being poured from a container immediately after shaking (A). One of the gel beads is highlighted to demonstrate the cell-binding agents bound to the surface. For demonstration purposes, the bead surface was engrafted with a CD34 antibody (the cell binding agent) incorporating a green fluorescent label. (B) shows how the surface of the highlighted bead is covered in green fluorescence indicating coverage with the CD34+ antibody. After mixing with bone marrow aspirate and removal of unbound cells, the surface of a bead is shown to be partially covered with CD34+ cells (red) (C).

(4) FIG. 3: This second example formulation depicts the calcium pyrophosphate star particles dispersed into the shear-thinning gel instead of gel beads in order to demonstrate the ability to deliver non-cell based therapeutic agents. The shear gel formulation in this example uses the same manufacture process as described in FIG. 2, but with Mn.sup.2+ ions (0.25M manganese sulfate in deionised water) used as the crosslinking agent. At rest the shear-thinning gel containing the star particles behaves as a monolith and maintains shape (A). (B) shows the a portion of the gel system shown in (A) loaded into a 10 mL syringe. Upon injection through the syringe, the star particle containing gel flows out of the syringe and can be extruded on to a surface (C,D). The extruded star particle containing gel thickens almost immediately after injection, remains in position even after inversion (E) and can be cleaved into portions (F,G). Only after repeated forceful impact against the work surface is the extruded gel moved around the dish (H).

(5) FIG. 4: Micro X-Ray Fluorescence maps of a 10 mm×6 mm portion (A) of an example formulation containing calcium pyrophosphate star particles, demonstrating even dispersion across a large area of gel. Locations containing calcium pyrophosphate were identified by mapping the presence of calcium (B) and phosphorous (C), with both maps overlaid (D). (E) shows how the star particles detected in (B) and (C) co-localise with the star-particles observed directly in the light microscope image of the same area (A).

SHEAR GEL PRODUCTION

(6) 18 ml 2% wt alginate in deionised water was added to a shear stirrer. This was warmed to 60° C. before shear force is applied via a rotor. 2 mL of 0.1M calcium chloride was added drop wise to the heated alginate solutions. The shear rate used was 450 s.sup.−1.

Alginate Bead Solution

(7) 4% wt alginate in deionised water at 60° C. was prepared. This was added drop wise through a hypodermic syringe into 1M calcium chloride in a beaker to form beads.

(8) Gel beads were mixed into shear thinning gel at 0.1 to 0.25 v/v.

(9) FIG. 1 shows the shear thinning properties of the formulation.

(10) FIG. 2 shows beads engrafted with CD34.sup.+ antibodies labelled with a green fluorescent label. (B) shows the beads glowing green with the labelled antibodies. (C) shows red labelled cells from bone aspirate attached to the beads vie the CD34.sup.+ antibodies. This shows enrichment of the cells by the antibodies or the beads.

Calcium Pyrophosphate Gels

(11) Shear gels were prepared as described above, but 0.25M MnSO.sub.4 was used as a cross linker.

(12) The particles of calcium phosphate were made by first preparing 300 mM aqueous solution of calcium chloride and a 150 mM aqueous solution of Sodium pyrophosphate decahydrate. Both solutions were adjust to pH 7 by addition of appropriate amounts of 12M hydrochloric acid and 1M sodium hydroxide while monitoring pH with a pH meter. Equal volumes of the calcium chloride solution and sodium pyrophosphate solutions were mixed together (200 mL of each in this case) under vigorous stirring for 1 minute at room temperature before the stirring was stopped and the reaction left alone for 1 hour. Within this time, a white precipitate appears to separate from the water and this precipitate has a fibrous appearance upon closer examination by eye. The water is decanted off and fresh deionised water is added to resuspend the precipitate and left overnight. The final product is then extracted from the water by vacuum filtration and dried under ambient conditions to form a powder of calcium pyrophosphate star-shaped particles. Further details of the production of such particles is described in WO 2008006204 A2, which is incorporated by reference in its entirety.

(13) At rest the shear thinning gel contains star shaped calcium pyrophosphate particles and behaves as a monolith and maintains shape (FIG. 3A). When loaded into a syringe (FIG. 3B) it can be extruded onto a surface (FIG. 3 C, D). This thickens almost immediately even after inversion (FIG. 3 E) and can be cleaved (FIG. 3E, F). Repeated impacts allowed the gel to move around the dish (FIG. 3H).

(14) Cells may be added to the composition. Antibiotics may also be used with composition

(15) The dispersion of calcium pyrophosphate star particles is shown in FIG. 4.