METHOD FOR COATING AND DRYING HETEROGENOUS STEM CELL DERIVED EXTRA-CELLULAR VESICLES

Abstract

Coated and Dried extracellular vesicles (EVs) represent an ideal method for preservation and increasing the shelf life-time of the invention making it ready to use on time and on variety of species overcoming the previous challenges on isolation and preservation and undesirable immune responses. The invention compromises a coated freeze dried stem cells derived EVs that are ready for use to Stimulate/accelerate healing of soft/hard tissues, can be reconstituted in multiple forms and shapes and stimulated by Laser. The nature of the invention is a heterogenous and/or Xenogenous EVs which can overcome the challenges of individual and/or species diseases and immune reactions.

Claims

1. A Lyophilized composition comprising stem cells (SCs) derived extra-cellular vesicles (EVs) obtained from heterogeneous donors.

2. A Lyophilized composition according to claim 1, wherein vesicles are coated by a protective substance to preserve their effectiveness.

3. A Lyophilized composition according to claim 1, wherein vesicles are further dried to improve storage time/conditions and facilitate the end-product formulation and transportation.

4. A Lyophilized composition according to claim 1, wherein vesicles may be used as is, mixed or added to other ingredient(s) to reach a specific biological and/or therapeutic effect(s), not limited to herbs, antibiotics, vitamins, or other pharmaceutical or biological ingredients. etc. to enhance/increase/add extra therapeutic/biological properties.

5. A Lyophilized composition according to claim 1, wherein vesicles combined with other polymers to produce biologically enhanced polymers.

6. A Lyophilized composition according to claim 1, wherein vesicles from heterogeneous donors are cross-used among different species (Xenogenously).

7. Method of preparation of Lyophilized composition according to claim 1, wherein the method comprises the following: (a) Bone marrow/adipose tissue is obtained from human, different animal species (dogs, cats, horses, ruminants, etc.). (b) SCs are isolated from bone marrow/adipose tissue in lab. (c) Isolated SCs are cultured for 3 to 4 weeks in incubators. (d) SCs are left to secrete EVs in a preconditioned media for 1 week. (e) Collection of EVs from the conditioned media using ultracentrifugation or fractionation.

8. Method of preparation of Lyophilized composition according to claim 7, wherein the method further comprise coating of extracellular vesicles (EVs) obtained/collected from a single and/or multiple sources “donor”, of a single and/or multiple species; by a protective substance to preserve their effectiveness and storage time using any of the following substances (e.g. and not limited to): (a) Polysaccharides, such as and not limited to starch, cellulose, gum, glycogen, gelatin, pectin, dextrin, alginate, chitosan. (b) Glycoprotein such as selectin, vegetable or mineral oils and fats such as lanolin and similar animal products such as egg white, milk or their natural or industrial derivatives. (c) Acids derived from sugars such as glycolic acid, tartaric, citric acid. (d) Liposomes (e) Nanoparticles and Nano carriers. (f) Natural or industrial plastics and polyether compounds such as Polyethylene glycol. (g) Therapeutic compositions: salt solutions/phosphate buffer saline, anti-inflammatories such as DMSO and the like.

9. Method of preparation of Lyophilized composition according to claim 7, wherein the method further comprise drying of the extracellular vesicles (EVs) obtained/collected from a single and/or multiple sources “donor”, of a single and/or multiple species; by means of (and not limited to) Lyophilization, Spray drying, Microwave assisted drying, Annealing, Desiccation and Vacuum dehydration.

10. Method of preparation according to claim 7 wherein the method comprises biological stimulation of the end-product (Kit) by laser. (a) The kit can be stimulated by low level laser exposure to increase the potency and proliferation. (b) The kit is exposed to wave low level laser with power density of 5.5 mW/cm.sup.2 and a wavelength of 635 nm for 20 min

11. Kit comprising composition according to claim 1, wherein the kit comprises Lyophilized composition can be formulated in different shapes for use according to the reconstitution method: (a) Reconstituted with little amount of sterile water or normal saline to give the shape of a viscous gel or ointment. (b) Further dilution to obtain a watery solution (drops or injection). (c) Watery solution packed in atomizer (spray or inhalation). (d) The diluted form can be electro-spun with other polymers to give different shapes and sizes like rods, threads, tablets, hydrogels, etc (for implantation). (e) The dried product is added to adhesive bandages as a wound/burn skin dressing.

12. Kit comprising composition according to claim 1, wherein the kit comprises Lyophilized composition collected from different donors (human or animals) is used among different recipients of the same species as a “Heterogenous” population. The “Heterogenous” population of the collected EVs is used also among different species “Xenogenous” e.g. From dogs to cats.

Description

DETAILED DESCRIPTION OF INVENTION

[0033] The invention compromises a method of preparation and preservation of Stem cell derived EVs to obtain a ready to use high grade concentrates of growth factors and cytokines to promote the healing of soft/hard tissues and can be used in different pharmaceutical shapes/forms with a long shelf life. And detailed as follows:

[0034] The product is prepared from a donor (human/animal) and is used in another recipient (human/animal) without any immune reaction. It can be used among different species and genera and is not limited as the previous preparations.

[0035] The EVs are loaded onto coating materials of different cyto-compatible natures.

[0036] The vesicles-loaded coating material is exposed to a drying process to extract water particles from it and convert it into solid/powder.

[0037] Preservation in room temperature without damage/reduced efficiency of the vesicles.

[0038] The final product can be prepared/shaped according to the required method of use (e.g. not limited to: fluids for injection by different routes or for oral intake in the form of tablets, pills, syrup, or the like). As well as local use as drops, ointments, or gels, or included in other compounds such as external dressings.

[0039] With the possibility of using it without an immune reaction, this guarantees the production of commercial quantities for use among humans and animals.

[0040] In addition, the coating and drying of the vesicles help to: (a) Protect the vesicles during the drying process, (b) Give the vesicles a degree of viscosity that enables them to hold together and bond with the tissue when treating wounds or diluting it according to the purpose of use, (c) When reconstituting the dried material, it returns to its original form immediately without significant change in the degree of viscosity, (d) Coating ensures that the outer cellular vesicles are preserved without a change in their therapeutic efficiency, (e) The ease of shaping the invention into different pharmaceutical forms (liquid/powder/tablets . . . ) that is appropriate to the place to be treated, (f) Drying makes it easy to produce, transport and use therapeutically and commercially.

Laboratory Experiments

[0041] Animal study (I) of reconstituted EVs on superficial wounds proved to: (a) Increase the speed of wound healing. (b) The healed tissue is of a better quality (less fibrous tissue), no scar. (c) Compared to commercial skin healing products it had a superior healing power regarding the healing time and quality. (d) in vitro analysis of the composition showed no significant change in physical or biological properties of the dried EVs.

[0042] Animal study (II) to evaluate the effect of Stem cell derived EVs in repair of induced chondral defects in a dog model showed that administration of EVs was effective on the functional and morphological recovery of the injured cartilage and could be exploited as a cell free therapeutic approach in regenerative medicine to achieve the restoration of chondral histomorphological picture within a period of 3 months with mature collagen fibers on histopathological evaluation on the contrary of the control joints that showed deterioration over time and defect filling with only fibrous tissue forming a fibrocartilage.

[0043] Side effects: No known side effects of the invention.

[0044] Method of exploitation (a) The dried product loaded with the EVs is reconstituted by distilled water or similar solvent. (b) The product returns to its original form immediately without any significant change and the required liquidity/viscosity. (c) The invention can be prepared and packaged in the form of tablets/capsules or similar to be taken orally or for the place to be treated (ointment/injection/oral/ . . . ).

Effectiveness

[0045] EVs perform the same work as the cells of origin and as efficiently as the freshly prepared one despite being dehydrated.

[0046] Stimulate/accelerate healing of soft/hard tissues for example, treat skin problems/wounds, heart problems, musculoskeletal degenerative disorders, hepatic and renal degenerative disorders, immune stimulation/modulation and tissue grafts.

[0047] Can be used for studies in vivo and in vitro for evaluation of biological experiments like cell signaling, proteomics, cellular proliferation etc.

[0048] Does not generate an immune reaction.

Expected Outcomes

[0049] Compared to the stem cell transplants, the invention holds the similar quality of healing.

[0050] Compared to conventional drugs, the invention can be used to achieve quicker and better healing quality of soft tissues/bones.

[0051] Absence of side effects such as, cell-toxicity, infection, immune reactions, or immune rejection.