Method for preparing a growth factors containing platelet releasate

Abstract

The present invention relates to a method for preparing a growth-factors containing platelet releasate from a fluid mammalian platelet concentrate, comprising the consecutive steps of subjecting the platelet concentrate to a pathogen reduction step to disrupt non-enveloped viruses; subjecting the platelet concentrate to an activation step to cause the platelets to release growth factors; recovering a fibrinogen depleted fluid platelet releasate; subjecting the fibrinogen depleted fluid platelet releasate to a second pathogen concentration reduction step to disrupt enveloped viruses; subjecting the platelet releasate to sterile filtering and recovering a filtrate liquid containing the growth factors. The platelet releasate obtained with the method of the present invention may be used as a therapeutic agent to enhance the proliferation of multi lineage cells in regenerative medicine and in the management of non healing wounds and resistant ulcers. The second indication is as a substitute to fetal bovine serum in in cell culture media.

Claims

1. A method for preparing a growth-factors containing platelet releasate from a fluid mammalian platelet concentrate having an initial platelet count, comprising the consecutive steps of a. subjecting the platelet concentrate to a first pathogen concentration reduction step with the purpose of disrupting both DNA and RNA of micro-organisms present in the platelet concentrate; b. subjecting the platelet concentrate to an activation step by contacting it with an activating agent, with the purpose of causing platelets to release at least part of alpha granules and growth factors present therein, thereby providing a fluid platelet releasate; c. subjecting the fluid platelet releasate to a fibrinogen concentration reduction step and recovering a fibrinogen depleted fluid platelet releasate; d. subjecting the fibrinogen depleted fluid platelet releasate to a second pathogen concentration reduction step with the purpose of disrupting enveloped viruses; e. subjecting the platelet releasate to sterile filtering and recovering from the filtering step a filtrate liquid containing the growth factors; f. dividing the filtrate liquid into individual portions which contain the releasate of about 2×10.sup.5−2×10.sup.7 platelets per cm.sup.3, based on the initial platelet count of the platelet concentrate, wherein the volume of the individual portion is adjusted such that each portion contains the releasate of approximately the same number of platelets; and g. subjecting the thus obtained individual portions of filtrate liquid to lyophilisation; wherein the growth-factors containing platelet releasate thereby obtained combines characteristics of being lyophilized in reasonable volumes which are suitable for immediate use without requiring further division into suitable aliquots, with ease of storage and reconstitution, and being fibrinogen depleted.

2. The method of claim 1, wherein the activating agent is a solution containing one or more compounds selected from the group of a calcium salt, thrombin, collagen, thromboxane A2 and adenosine diphosphate (ADP).

3. The method according to claim 1, wherein the first pathogen concentration reduction step comprises incubating the platelet concentrate with a photochemical active agent and exposing the platelet concentrate to UV irradiation.

4. The method according to claim 3, wherein the UV irradiation is UVA irradiation.

5. The method according to claim 3, wherein the photochemical active agent is a psoralen.

6. The method of claim 1, wherein the second pathogen concentration reduction step comprises incubating the platelet releasate with a solvent, a detergent, or a combination thereof; followed by removing the solvent, the detergent or the combination thereof; and recovering the platelet releasate depleted for the solvent, the detergent, or the combination thereof.

7. The method of claim 6, wherein the solvent for incubating the platelet releasate is selected from the group consisting of dialkylphosphates and trialkylphosphates.

8. The method of claim 6, wherein the detergent is one or more selected from the group consisting of polyoxyethylene derivatives of fatty acids, partial esters of sorbitol anhydrides, non-ionic detergents, sodium deoxycholate and sulfobetaines.

9. The method according to claim 6, wherein the solvent, the detergent, or the combination thereof is removed from the platelet releasate by oil extraction.

10. The method according to claim 9, wherein the oil extraction is carried out in the presence of an amount of oil which ranges from 2 to 20 weight %, based on the total weight of the mixture of the platelet releasate and the solvent, the detergent, or the combination thereof.

11. The method according to claim 1, further comprising at least one step of centrifugation of the platelet releasate, after removal of the solvent, the detergent, or the combination thereof in the second pathogen concentration reduction step.

12. The method according to claim 1, wherein human albumin is supplied to the filtrate liquid obtained in step e, in advance of subjecting the filtrate liquid to lyophilisation.

13. The method according to claim 1, wherein in advance of subjecting the platelet concentrate to the activation step b, the platelet concentrate is subjected to a leukocyte inactivation step.

14. The method according to claim 1, wherein the mammalian platelet concentrate is rich in growth factors from human platelets or horse platelets.

15. The method according to claim 1, wherein the mammalian platelet concentrate comprises a pool of platelet concentrates which originate from different subjects.

16. The method of claim 2, wherein the activating agent is a thrombin containing solution.

17. The method of claim 4, wherein the UVA irradiation has an energy density between 1 and 10 J/cm.sup.2.

18. The method of claim 17, wherein the UVA irradiation has an energy density of 3 J/cm.sup.2.

19. The method according to claim 5, wherein the photochemical active agent is amotosalen or riboflavin.

20. The method of claim 7, wherein the solvent for incubating the platelet releasate concentrate is tri-n-butylphosphate.

21. The method of claim 8, wherein the detergent is Triton X-45, Triton X-100 or Tween 80.

22. The method according to claim 10, wherein the oil extraction is carried out in the presence of an amount of oil which ranges from 5 to 15 weight %, based on the total weight of the mixture of the platelet releasate and the solvent and/or detergent.

23. The method according to claim 22, wherein the oil extraction is carried out in the presence of an amount of oil which ranges from 5 to 10 weight %, based on the total weight of the mixture of the platelet releasate and the solvent and/or detergent.

24. The method of claim 1, wherein the filtrate liquid is divided in individual portions which contain the releasate of about 2×10.sup.6 platelets per cm.sup.3, based on the initial platelet count of the platelet concentrate.

25. A method for at least enhancing proliferation of multilineage cells, promoting wound healing, or ulcer healing; comprising treating a subject in need thereof with an effective amount of the growth-factors containing platelet releasate obtained with the method of claim 1.

26. A cell culture medium for growth of at least stem cells, fibroblasts or dendritic cells; comprising a growth-factors containing platelet releasate obtained with the method of claim 1.

27. A formulation containing the growth-factors containing platelet releasate obtained with the method according to claim 1, which is one or more selected from the group consisting of an aerosol, a treatment fluid, a spray, a mist, a lotion, a cream, an ointment, a gel, a gum, a bandage, a dermal patch, and a plaster.

Description

Example 1

(1) A human was connected to a pheresis machine (Haemonetics_MCS+9000.sup.a) so as to obtain a platelet rich concentrate, with platelet count 3-5 times the initial platelet count. The platelet rich concentrate was incubated with Riboflavin and irradiated with UV rays by the MIRASOL™ system as a first step of pathogen reduction. Thereafter, sterile Thrombin.sup.b was added in a suitable concentration of 500 units/cmm to activate the platelets in said concentrate. This gave rise to subsequent release from platelet granules of supra physiological doses of growth factors and cytokines. In addition, activated platelets released the alpha granules content of fibrinogen which is activated by thrombin to form an insoluble clot. When centrifuged, the thrombin treated platelet concentrate separated into a supernatant comprising a clear light red liquid or fluid that consists essentially of lysed platelets, contents of which are supra physiological doses of growth factors released from platelets granules, and a deposit of insoluble fibrin clot. The supernatant was transferred into a new container for further steps of preparation. The platelet releasate was treated with a solvent and detergent system (0.3% TNBP.sup.c and 1% tween 20.sup.d), for 1 hour at 31° C. This step is capable of disrupting enveloped viruses (mainly HBC, HCV, and HIV). The solvent and the detergent are there after removed by three consecutive steps of vegetable oil extraction. Sterile castor oil (7.5% of total volume) is added to platelet releasate and mixture is subjected to shaking on a blood mixer for 15 minutes then the container is suspended in an inverted position for 10 minutes to obtain an upper oil phase and a lower platelet lystae phase, which is separated by gravity from the upper phase. This step is repeated for two other times, then the platelet releasate is centrifuged for 20 minutes at 4° C. and 1500 g, in an inverted position to separate any remanants of oil in an upper phase and the platelet releasate in a lower phase that is obtained by gravity

(2) Then, the platelet releasate is subjected to a step of Sterile filtering to ensure removal of any bacterial contaminants and then the liquid or fluid filtrate is dispensed in predetermined volumes, (adjusted according to initial platelet counts of concentrate to ensure an equivalent of around 1 million platelets per cm.sup.3) in glass vials and lyophilizing such filtrate which consists mainly of platelet derived growth factors.

Example 2

(3) The platelet releasate obtained in example 1 was used to achieve facial rejuvenation. One vial which originated from 2×10.sup.6 platelets was injected on each side of the face of a women in the chin, cheek and nasolabial fold. The redness, pain and swelling could be significantly reduced. The regenerative effect lasts for at least 6 months.

Comparative Experiment

(4) A volume which contained 2×10.sup.6 autologous platelets was injected on each side of the face of a women, suffering from the same defects as in example 2. The redness, pain and swelling could be reduced to a limited extent only.

Example 3

(5) The platelet releasate obtained in example 1 was used to achieve pain relief in low back pain management. Two vials which originated from 2×10.sup.6 platelets were injected in the lower part of the back. The pain could be significantly reduced. The treatment was repeated a week later, by injection of one vial. The regenerative effect lasted for several months.

Example 4

(6) The platelet releasate obtained in example 1 was incorporated into a collagen sodium alginate hydrogel wound dressing, and was applied to a diabetic ulcers that could not be healed for more than 6 months. Within a few days significant wound healing could be achieved.

Example 5

(7) A platelet releasate was prepared according to the method of example 1, this time using horse blood. Two vials which each originated from 2×10.sup.6 platelets were injected in injected into an injured muscle of a horse. The treatment was repeated after a week. The muscle healed with no remaining injury.