Multicellular lay-up process

Abstract

Disclosed herein is a multicellular lay-up process. The process comprises the steps of: a) forming a core material, b) forming a capsule material, c) encapsulating the core with the capsule material, d) adding the capsule to a substrate, and e) exposing the capsule to at least one bioactivating agent.

Claims

1. A multicellular lay-up process, the process comprising the steps of: a) forming a core material, b) forming a capsule material, c) encapsulating the core with the capsule material, d) adding the capsule to a substrate, and e) exposing the capsule to at least one bioactivating agent, wherein the capsule material comprises a water soluble polymer, a fibrous component, and a solvent, wherein lengths and sizes of the fibrous component are maintained within a predetermined range, and wherein the predetermined range is selected to achieve a desired capsule viscosity.

2. The process according to claim 1, wherein the method comprises forming a plurality of capsules, adding a plurality of capsules to a substrate and exposing the plurality of capsules to at least one bioactivating agent.

3. The process according to claim 1, wherein the core comprises one or more mammalian cells, cell culture medium and a hydrogel.

4. The process according to claim 3, wherein the cells are stem cells.

5. The process according to claim 3, wherein the cell culture medium comprises selective growth medium, growth factor or buffer.

6. The process according to claim 3, wherein the hydrogel comprises any of gelatin, polyethylene glycol, glycerol, alginate, dextran-40, trehalose, or DMSO.

7. The process according to claim 3, wherein the core further comprises at least one bioactivating agent selected from the group consisting of growth factors, growth inhibitors, antimicrobial agents or anti-inflammatory agents.

8. The process according to claim 1, wherein the step of forming the core comprises combining one or more mammalian cells, at least one bioactivating agent, cell culture medium and a hydrogel.

9. The process according to claim 1, wherein the water soluble polymer comprises polyethylene oxide and/or polyethylene glycol.

10. The process according to claim 1, wherein the fibrous component comprises cryomilled bioactive nano fibres, cryosonic milled fibres, or lithographic cut fibres.

11. The process according to claim 10, wherein the fibres comprise natural and/or synthetic polymers.

12. The process according to claim 11, wherein the natural polymers comprise cross-linked collagen or cross-linked hyaluronic acid.

13. The process according to claim 11, wherein the synthetic polymers comprise polylactide glycolic acids, poly lactic acid, poly glycolic acid or polycapralactone.

14. The process according to claim 10, wherein the fibrous component is infused with an enzyme.

15. The process according to claim 10, wherein the fibrous component is infused with an electromagnetic absorber.

16. The process according to claim 1, wherein the solvent comprises any of water or organic solvents.

17. The process according to claim 1, wherein the capsule material further comprises at least one bioactivating agent selected from the group consisting of group consisting of, growth factors, growth inhibitors, antimicrobial agents or anti-inflammatory agents.

18. The process according to claim 1, wherein the step of encapsulating the core with the capsule material comprises electrospraying both the formed cores and the capsule material concentrically in a cryogenically cooled drop tower.

19. The process according to claim 1, wherein the step of adding the capsule to the substrate is performed in an additive manufacturing process.

20. The process according to claim 19, wherein the additive manufacturing process is any of high speed sintering, ink jet printing, poly jet printing, spraying, high resolution deposition, spraying, syringe dispensing, near field electrospray or aero-sol jetting.

21. The process according to claim 19, wherein the adding step further comprises the deposition of a barrier on the substrate.

22. The process according to claim 21, wherein the barrier comprises dry hydrophobic particles.

23. The process according to claim 22, wherein the hydrophobic particles comprise PTFE particles.

24. The process according to claim 22, wherein the deposition of dry hydrophobic particles on the substrate is by any of high speed sintering, ink jet printing, poly jet printing, spraying, high resolution deposition, spraying, syringe dispensing, near field electrospray or aero-sol jetting.

25. The process according to claim 1, wherein the step of exposing the capsule to at least one bioactivating agent comprises irrigating the capsules with an aqueous solution comprising one or more bioactivating agents.

26. The process according to claim 1, wherein the exposing is performed by any of high speed sintering, ink jet printing, poly jet printing, spraying, high resolution deposition or aerosol jetting.

27. The process according to claim 25, wherein the one or more bioactivating agents are selected from the group consisting of group consisting of growth factors, growth inhibitors, antimicrobial agents or anti-inflammatory agents.

28. The process according to claim 1, wherein steps d) and e) are repeated consecutively in order to lay-up a three dimensional array of capsules.

29. The process according to claim 1, wherein capsules formed are between 20 μm and 50 μm in diameter.

30. The process according to claim 1, wherein a plurality of capsules comprising cell types programmed to produce a first tissue type are laid down on the substrate adjacent to a plurality of capsules comprising cell types programmed to produce a second tissue type.

31. The process according to claim 1, wherein the core, capsule material or fibrous component further comprises a near infrared absorber.

32. A multicellular lay-up process, the process comprising the steps of: a) forming a core material, b) forming a capsule material, c) encapsulating the core with the capsule material, d) adding the capsule to a substrate, and e) exposing the capsule to at least one bioactivating agent, wherein the capsule material comprises a water soluble polymer, a fibrous component, and a solvent, wherein the core comprises one or more mammalian cells, cell culture medium and a hydrogel, wherein the fibrous component comprises cryomilled bioactive nano fibres, and wherein at least one of the cryomilled bioactive nano fibres resides entirely within the capsule material.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The invention will now be described by way of example and/or illustration only with reference to the accompanying drawings in which:

(2) FIG. 1 is a cross-section of an embodiment of the capsule manufactured according to an embodiment of the method of the invention.

(3) FIG. 2 shows and example of the electro spinning set up used in the invention.

(4) FIG. 3 shows an SEM image of the fibre mat.

(5) FIG. 4 shows and SEM image of the cryomilled fibre mat.

(6) FIG. 5 shows a schematic of an embodiment of the method of forming the capsules around the core.

(7) FIG. 6 shows a schematic of an embodiment of the additive layer manufacturing method.

(8) FIG. 7 shows a schematic of an embodiment of the method of addition of barrier materials to the substrate.

(9) FIG. 8 shows the irrigation of thawed cryo capsules with solutions.

(10) FIG. 9 shows a cross section of the results of an embodiment of the method where steps D and e are repeated.

(11) FIG. 10 shows the resultant tissue/organ bioreactor

(12) FIG. 11 shows the tissue/organ recovery after hydrophobic particle removal.

DETAILED DESCRIPTION OF THE ILLUSTRATED EMBODIMENT

(13) An embodiment of the invention will now be described by way of example.

(14) Core Formation

(15) A core material was formed by mixture of stems cells (1), with a cell culture media, cryo protectants and bioactive agents (2). The mixing was performed under sterile conditions. See FIG. 1 for a representation of the core within the capsule 3.

(16) Generation of Nano Fibre Segments

(17) Using a typical electro spinning apparatus (see FIG. 2) a non-woven mat of poly lactic-co-glycolic acid [PLGA] was produced (FIG. 3). As an example an electrospinning solution of 6 wt % PLGA polymer (Purac) dissolved in Hexafluoroisopropanol (HFIP) was mixed with an aqueous solution of 20 microgrammes of lyophilized growth factor in 333 microlitres of 5 mM TRIS (pH 7.6) containing 0.1% Bovine Serum Albumin). The solution was loaded into poly propylene syringe which is connected to a stainless steel 21 Gauge blunt end tube by PTFE tubing. The tube was connected to a high voltage supply. The temperature of the electrospinning solution was maintained below 37 C. The environment between the electrospinning needle electrospinning solution was controlled so that the relative humidity and the ambient temperature did not vary. Typical values were an RH of 60% and a temperature of 25 C. A rotating collecting drum which had a conductive surface was positioned at a distance of 300 mm from the open end of the stainless steel tube. The surface of the drum was biased at a voltage between 0 kV and −12.5 kV. The stainless steel tube was biased at 10 kV to 30 kV. The electrospinning solution was pumped at a controlled rate (1 mL/hour) through the tube. Electrostatic forces acting on the solution emerging from the end of the tube caused a cone and jet to form. The rheological properties and the molecular weight of the PLGA resulted in entanglement as solvent was lost from the surface of the solution. This resulted in fibre formation. Electrostatic charge density increased as the fibre moved toward the collector drum. The electrostatic forces arising caused elongational extension of the fibre such that micron and nanoscale fibres could be produced. There was a random flight of the fibre between the tube and the collector and this resulted in lay down of a random nanofibre mat of the rotating collector. After a defined time the mat was removed and allowed to dry to ensure all solvents were removed. Then a PEG and/or PEG solution was applied and allowed to dry. The weight ration of the PEG and/or PEG polymer to the weight of nanofibre was controlled. The dry mat was then cryomilled to create particles infused with short fibres (FIG. 4).

(18) Capsule Material Generation

(19) The capsule material was then formed by dissolving the particles produced in water to create the capsule material formulation. For a PEO polymer with a Molecular Weight of 2,000,000 of PEO a 2 wt % ratio of particles to water was used.

(20) Encapsulation of the core with the capsule material.

(21) Using an electro spray apparatus the core material 1 and the capsule material 3 were released/sprayed into a container over liquid nitrogen vapour. The electro spray apparatus had a concentric delivery tube 4 so that the two materials were delivered together (see FIG. 5). Electrostatics and solvent loss created a core-shell particle structure prior to the capsule passing through a liquid nitrogen vapour. What resulted was a cryo biocapsule powder. The powder of capsules were maintained in a cryo preservation state in liquid nitrogen vapour for storage purposes

(22) Adding Capsule to the Substrate (FIG. 6)

(23) In this step a fluidised bed 5 of the biocapsule power was created by suspending the powder in forced flow liquid nitrogen vapour. Samples of the vapour which contained some capsules were then extracted and combined with warm air using a venturi. The warm air caused controlled heating in the delivery tube of the capsules to ensure delivery above 0 C. Using a smooth bore PTFE tubing and PTFE needle, the capsules were then deposited on a temperature controlled PTFE tray 6 and allowed to thaw. Two sets of capsules were deposited both in single layers on distinct areas of the try. In this step, but using a different delivery system, the PTFE capsules 8 (FIG. 7) were added to the tray around the capsules 10.

(24) Exposing the Capsules to the Bioactivating Agents (FIG. 8).

(25) Using a third delivery system a bioactive agent A in an aqueous solution was irrigated over the first group of capsules and an aqueous solution with bioactive agent B was irrigated over the second group of capsules. The process of adding the capsules to the substrate and adding the PTFE and adding the bioactive agent was repeated (FIG. 9) until stacks 12 of the two sets of capsules were created.

(26) The PTFE particles were then removed and culture medium added. Then culture medium was added and the capsules disintegrated leaving just the two groups of differentially activated cells.

(27) See FIG. 10 for the resultant tissue/organ bioreactor, which is then cultured to produce the tissue/organs required (FIG. 11).