WO2023052954A1

WO2023052954A1 - Method of preparing a solution for activating platelets

Info

Publication number: WO2023052954A1
Application number: PCT/IB2022/059104
Authority: WO
Inventors: Alexandra Grace MISZEWSKI; Judey PRETORIUS
Original assignee: Miszewski Alexandra Grace
Priority date: 2021-09-30
Filing date: 2022-09-26
Publication date: 2023-04-06

Abstract

The invention relates to a method of preparing a solution in a container for activating platelets and/or extracting growth factors from the platelets in the container for use in regenerative medicine treatment, said method including adding a first anti-coagulant being a sodium citrate solution and adding a preparation of recombinant collagen and adding a second anti-coagulant; and adding a Sodium Chloride solution. The invention further relates to a method of activating platelets and/or extracting growth factors from the platelets in blood, wherein the method includes introducing a container having the solution of the first aspect of the invention and adding blood to the container and then centrifuging the container and lastly extracting the processed or part of the processed blood.

Description

METHOD OF PREPARING A SOLUTION FOR ACTIVATING PLATELETS

FIELD OF THE INVENTION

This invention relates to a method of preparing a solution for a tube. In particular, this invention relates to a method of preparing a solution for a tube for facilitating the isolation of blood platelets and extraction of growth factors from the blood platelets along with the stabilization of the growth factors after extraction has occurred. The invention further relates to the use of said extracted product as a regenerative medicine treatment, in particular for “growth-factor rich” or “cytokine-rich” regenerative medicine treatment

BACKGROUND TO THE INVENTION

Platelets, also called thrombocytes, are tiny fragments of cells that are essential for normal blood clotting and other necessary growth healing functions. They are formed from very large cells called megakaryocytes in the bone marrow and are released into the blood to circulate. It is the activation of these platelets that plays a key role in the natural healing process of a body of a mammal. By isolating the platelets from the blood, the platelets can be activated allowing the platelets to release growth factors that stimulate and increase the number of reparative cells the body of the mammal produces.

There are many known solutions for tubes which are used to facilitate the isolation of blood platelets from blood in a blood sample to allow activation of the blood platelets for use as a regenerative medicine treatment, However, these known solutions do not activate the platelets or extract the growth factors from the platelets in a single method, within a single tube, as conventional systems only achieve activation of platelets in a separate second step or method outside of the tube.

Embodiments of the invention thus aim to address the issues identified above, at least to some extent.

SUMMARY OF THE INVENTION

According to the invention, there is provided for a method of preparing a solution in a container for activating platelets and/or extracting growth factors from the platelets in the container for use in regenerative medicine treatment, said method including: adding a first anti-coagulant being a sodium citrate solution; adding a preparation of recombinant collagen; adding a second anti-coagulant; and adding a Sodium Chloride solution.

In one embodiment the sodium citrate solution may be sparged with filtered nitrogen.

In another embodiment, the pH of the sodium citrate solution may be adjusted.

In still another embodiment, the sodium citrate solution may be filtered.

In yet another embodiment, the sodium citrate solution may be sterilized.

Furthermore, the recombinant collagen may be a freeze-dried preparation of recombinant collagen to form a sodium citrate collagen solution.

In one embodiment, the second anti-coagulant may be a pre-coated layered anticoagulant acid citrate dextrose (ACD-A) solution.

Still further, the sodium citrate solution may be sparged with filtered nitrogen for a period in the range of 10 minutes to 25 minutes, preferably being a period of 18 minutes.

Yet still further, the sodium citrate solution may have a volume in the range of 0.5 ml to 1 .2 ml, preferably having a volume of 1 ml. In an ideal embodiment, the container may be in the form of a tube which complies to international compendia standards, wherein the tube is a glass tube having siliconized coating on the inner walls thereof, and wherein the degree and/or amount of siliconized coating is controlled to manipulate the roughness of the inner wall surface of the tube to smoothen or reduce a degree of the roughness of the inner walls of the tube, to such an extent, to still enable slight activation during centrifugation, whilst preventing excessive activation thereby enabling optimum lysation of the platelets.

Still further where sealing means and vacuum may be applied to the container and wherein the vacuum has a draw volume between 1 ml and 7ml. Ideally, wherein the vacuum is a 3ml draw volume.

In still another embodiment, wherein Gamma Radiation may be applied as a final step of preparing the solution.

In yet another embodiment, wherein the pH of the sodium citrate solution may be adjusted by the addition of sodium hydroxide solution, wherein the sodium hydroxide solution may have a ratio of 10% sodium hydroxide to 90% water, and wherein the pH of the sodium citrate solution may be in the range of 7.0 to 8.0. Ideally, wherein the pH may be a pH of 7.2.

In one embodiment, the filter used in filtering the sodium citrate solution may be in the form of a 0.2pm filter, wherein the filter may further include a laminar flow hood and a UV light arrangement to facilitate filtering the sodium citrate solution.

Further, the conductivity of the sodium citrate solution may be measured, wherein the conductivity may be less than 1 .1 ps.

Moreover, the specific density of the sodium citrate solution may be measured, and wherein the specific density may be at least 1 g/cm³. Still further, the sodium citrate solution may be sterilized using steam for a period in the range of 10 minutes to 25 minutes before introducing the preparation of recombinant collagen. Ideally, wherein sterilization may be for a period of 18 minutes, wherein the steam may have a temperature in the range of 120 °C to 130 °C. Ideally, wherein the steam may be 125 °C.

In one embodiment, the freeze-dried preparation of recombinant collagen may have a volume in the range of 100 pl to 200 pl. Ideally, wherein the volume is 200 pl.

The recombinant collagen may be in the form of a biosynthetic recombinant collagen, wherein the biosynthetic recombinant collagen may have a single chain structure with fixed molecular weight in the range of 10 KDa to 100 KDa. Ideally, having a fixed molecular weight of 55 KDa, wherein the single chain structure is in the form of a modified G-X-Y characteristic sequence which contains no immunogenic end.

Further, the solution in the tube may be vortexed for a period in the range of 3 minutes to 5 minutes, wherein the solution may be vortexed for a period of 3 minutes.

Still further, wherein the solution in the container may be placed in a shaker for a period in the range of 8 hours to 10 hours.

Yet still further, wherein the volume of the sodium citrate collagen solution in the container may be placed in a shaker for a period of 8 hours, at room temperature, until a homogenous mixture is obtained.

In another embodiment, wherein the container with the volume of the solution may be placed in an oven with a temperature in the range of 55 °C to 65 °C, wherein the container with the volume of the sodium citrate collagen solution may be placed in an oven with a temperature of 65 °C. In one embodiment, wherein the container may be placed in the oven for a period in the range of 8 hours to 12 hours, wherein ideally the container is placed in the oven for 12 hours.

In yet another embodiment, wherein a volume of 0.2ml up to 1.0 ml of a Sodium Chloride solution having a concentration rage of 0.5% up to 30% may be added.

Still further, wherein the Sodium Chloride solution may be a 0.5ml Sodium Chloride solution, said Sodium Chloride solution may have a 0.9% concentration.

In an ideal embodiment, wherein the method may be a single continuous method, within a single container.

According to a second aspect of the invention, a method of activating platelets and/or extracting growth factors from the platelets in blood, said method may include:

Introducing a container having the solution of the first aspect of the invention;

Adding blood to the container;

Centrifuging the container; and

Extracting the processed or part of the processed blood.

In one embodiment, wherein extracting the processed or part of the processed blood may be the extraction of growth factors.

According to a third aspect of the invention, processed blood, may be obtained from the method of the second aspect of the invention, for use in regenerative medicine treatment, and wherein ideally the processed blood may be used in growth factor or cytokine rich regenerative medicine treatment.

The blood according to the second aspect of this invention, wherein the blood may be human blood. The blood according to the second aspect of this invention, wherein the blood may be animal blood

The blood according to the second aspect of this invention, wherein the blood may be other vertebrate blood.

DETAILED DESCRIPTION OF THE INVENTION

The following description is provided as an enabling teaching of the invention, is illustrative of principles associated with the invention and is not intended to limit the scope of the invention. Changes may be made to the embodiment/s depicted and described, while still attaining results of the present invention and/or without departing from the scope of the invention. Furthermore, it will be understood that some results or advantages of the present invention may be attained by selecting some of the features of the present invention without utilising other features. Accordingly, those skilled in the art will recognise that modifications and adaptations to the present invention may be possible and may even be desirable in certain circumstances, and may form part of the present invention.

Figure 1 is a schematic of a method of preparing a solution for a tube in accordance with the present invention.

Referring now to the drawing reference numeral 10 refers generally to a method of preparing a solution for a tube, which method includes mixing sodium citrate with water to form a sodium citrate solution 12, sparging the sodium citrate solution 12 with filtered nitrogen, adding a sodium hydroxide solution to the sodium citrate solution 12 to adjust the pH of the sodium citrate solution 12, filtering the sodium citrate solution 12 with a filter 14, sterilizing the sodium citrate solution 12, and mixing the sodium citrate solution 12 with a freeze-dried preparation of recombinant collagen 16 to form a sodium citrate collagen solution 18.

A pre-coated layered anti-coagulant acid citrate dextrose (ACD-A) solution (not shown) is added to the sodium citrate solution 12.

The sodium citrate solution 12 is sparged with filtered nitrogen for a period in the range of 10 minutes to 25 minutes, typically being a period of 18 minutes. The sodium citrate solution 12 is sparged into a tube (not shown). The sodium citrate solution 12 has a volume in the range of 0.5 ml to 1 .2 ml, typically having a volume of 1 ml.

The tube (not shown) is in the form of a glass tube which complies to international compendia standards.

The sodium hydroxide solution has a ratio of 10% sodium hydroxide solution to 90% water.

The pH of the sodium citrate solution 12 is in the range of 7.0 to 8.0, typically having a pH of 7.2.

The filter 14 in the form of a 0.2pm filter. The filter 14 further includes a laminar flow hood and a UV light arrangement to facilitate filtering the sodium citrate solution 12.

The conductivity of the sodium citrate solution 14 is measured and should be less than 1 .1 ps. The specific density of the sodium citrate solution 12 is measured and should be at least 1 g/cm3.

The sodium citrate solution 12 is sterilized using steam for a period in the range of 10 minutes to 25 minutes, typically being a period of 18 minutes. The steam has a temperature in the range of 120 °C to 130 °C, typically having a temperature of 125 °C.

The freeze-dried preparation of recombinant collagen 16 has a volume in the range of 100 pl to 200 pl, typically having a volume of 200 pl. The recombinant collagen 16 is in the form of a biosynthetic recombinant collagen. In particular, the biosynthetic recombinant collagen 16 has a single chain structure with fixed molecular weight in the range of 10 KDa to 100 KDa, typically having a fixed molecular weight of 55 KDa. The single chain structure is in the form of a modified G-X-Y characteristic sequence which contains no immunogenic end. It is to be appreciated that that the single chain results in a low molecular weight of recombinant collagen compared to the high molecular weight of normal collagen which has a triple chain.

Furthermore, it is to be appreciated that the low and constant molecular weight of the biosynthetic recombinant collagen ensures consistent rates of transdermal absorption through mucus membranes and to enhance the process of angiogenesis.

A volume of the sodium citrate collagen solution 18 is added to the tube (not shown) via sparging. The volume is in range of 0.5 ml to 1.2 ml, typically being 0.5 ml. The tube (not shown) with the volume of the sodium citrate collagen solution is vortexed for a period in the range of 3 minutes to 5 minutes, typically being a period of 3 minutes. Furthermore, the tube (not shown) with the volume of the sodium citrate collagen solution is placed in a shaker for a period in the range of 8 hours to 10 hours, typically being a period of 8 hours, at room temperature until a homogenous mixture is obtained. Yet furthermore, the tube (not shown) with the volume of the sodium citrate collagen solution 18 is placed an oven with a temperature in the range of 55 °C to 65 °C, typically having a temperature of 65 °C, for a period in the range of 8 hours to 12 hours, typically being a period of 12 hours.

A further volume in range of 0.5 ml to 1 .2 ml, typically being 1 ml, of the sodium citrate collagen solution 18 is added to the tube (not shown), via sparging, after the tube (not shown) is removed from the oven.

Furthermore, the tube (not shown) with the volume of the sodium citrate collagen solution 18 is placed in a shaker for a period in the range of 12 hours to 24 hours, typically being a period of 16 hours at room temperature.

A further volume in range of 20 pl to 100 pl of the sodium citrate solution 16 is added to the tube (not shown), via sparging. A further volume of 0.5ml Sodium Chloride (not shown) of a 0.9% concentration may be added to the tube as a final step.

Furthermore, vacuum (not shown) is used. The vacuum is applied after production. It only takes a few seconds as the tube and the stopper is put in the vacuum machine and then the tube stopper is placed into the tube inside the machine to “capture” the vacuum.

Moreover, the final step is sterilization wherein Gamma radiation is applied to the container, wherein a dose of 5KGY up to 25KGY, within an ideal dose of 10KGY.

In use, 6 to 8 ml of blood is drawn from a vein (not shown) of a mammal (not shown) and extruded into the tube (not shown) and into the solution of the instant method 18. Ideally under vacuum and is performed at ambient temperatures. The blood and solution 18 within the tube (not shown) is then spun in a centrifuge for 12 to 18 minutes between 2000 to 3000 revolutions per minute (RPM). During this process the solution 18 facilitates the isolation of blood platelets from the blood and extracts growth factors from the blood platelets. Furthermore, the sodium citrate collagen solution 18 stabilizes the growth factors after extraction has occurred.

The inventor believes that the method of preparing a solution for a tube in accordance with the present invention is advantageous in that it not only provides a solution which facilitates the isolation of blood platelets but also facilitates the extraction of growth factors from the blood platelets while minimising the pro inflammatory cytokines and stabilising the growth factors.

Furthermore, the method is advantageous in that it stabilizes the solution throughout preparation thereof allowing the solution to be kept at room temperature without the denaturing of proteins within the solution occurring. Furthermore, it is advantageous in that the method, and the solution prepared by the method, are easy to use.

Yet another advantage of the invention is that the sparging of the sodium citrate solution and the sodium citrate collagen solution into the tube creates layers on an inner surface of the test tube. These layers create a greater surface area for more blood to get lysed thereby facilitating the extraction of growth factors, as well as to create a homogenous mixture of growth factors.

Citation or discussion of a reference herein shall not be construed as an admission that such is prior art to the present invention.

Claims

1 . A method of preparing a solution in a container for activating platelets and/or extracting growth factors from the platelets in the container for use in regenerative medicine treatment, said method including:

Adding a first anti-coagulant being a sodium citrate solution;

Adding a preparation of recombinant collagen;

Adding a second anti-coagulant; and

Adding a Sodium Chloride solution.

2. The method of claim 1 , wherein the sodium citrate solution is sparged with filtered nitrogen

3. The method of claim 1 , wherein the sodium citrate solution’s pH is adjusted.

4. The method of claim 1 , wherein the sodium citrate solution is filtered.

5. The method of claim 1 , wherein the sodium citrate solution is sterilized.

6. The method of claim 1 , wherein the recombinant collagen is a freeze-dried preparation of recombinant collagen to form a sodium citrate collagen solution.

7. The method of claim 1 , wherein the second anti-coagulant is a pre-coated layered anti-coagulant acid citrate dextrose (ACD-A) solution.

8. The method of claim 2, wherein the sodium citrate solution is sparged with filtered nitrogen for a period in the range of 10 minutes to 25 minutes, preferably being a period of 18 minutes.

9. The method of claim 1 , wherein the sodium citrate solution has a volume in the range of 0.5 ml to 1 .2 ml, preferably having a volume of 1 ml.

10. The method of claim 1 , wherein the container is in the form of a tube which complies to international compendia standards.

1 1. The method of claim 10, wherein the tube is a glass tube having siliconized coating on the inner walls thereof.

12. The method of claim 11 , wherein the degree and/or amount of siliconized coating is controlled to manipulate the roughness of the inner wall surface of the tube to smoothen or reduce a degree of the roughness of the inner walls of the tube, to such an extent, to still enable slight activation during centrifugation, whilst preventing excessive activation thereby enabling optimum lysation of the platelets.

13. The method of claim 1 , wherein sealing means and vacuum is applied to the container.

14. The method of claim 13, wherein the vacuum has a draw volume between 1 ml and 7ml.

15. The method of claim 14 wherein the vacuum is a 3ml draw volume.

16. The method of claim 13, wherein Gamma Radiation is applied as a final step.

17. The method of claim 3, wherein the pH of the sodium citrate solution is adjusted by the addition of sodium hydroxide solution.

18. The method of claim 17, wherein the sodium hydroxide solution has a ratio of 10% sodium hydroxide to 90% water.

19. The method of claim 3, wherein the pH of the sodium citrate solution is in the range of 7.0 to 8.0.

20. The method of claim 3, wherein the pH is a pH of 7.2.

21. The method of claim 4, wherein the filter for use in filtering the sodium citrate solution is in the form of a 0.2pm filter.

22. The method of claim 21 , wherein the filter further includes a laminar flow hood and a UV light arrangement to facilitate filtering the sodium citrate solution.

23. The method of claim 1 , wherein the conductivity of the sodium citrate solution is measured.

24. The method of claim 23, wherein the conductivity is less than 1.1 ps.

25. The method of claim 1 , wherein the specific density of the sodium citrate solution is measured.

26. The method of claim 25, wherein the specific density is at least 1g/cm³.

27. The method of claim 5, wherein the sodium citrate solution is sterilized using steam for a period in the range of 10 minutes to 25 minutes before introducing the preparation of recombinant collagen.

28. The method of claim 27, wherein sterilization is for a period of 18 minutes.

29. The method of claim 27, wherein the steam has a temperature in the range of 120 °C to 130 °C.

30. The method of claim 27, wherein the steam is 125 °C.

31. The method of claim 6, wherein the freeze-dried preparation of recombinant collagen has a volume in the range of 100 pl to 200 pl.

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32. The method of claim 31 , wherein the volume is 200 pl.

33. The method of claim 1 , wherein the recombinant collagen is in the form of a biosynthetic recombinant collagen.

34. The method of claim 33, wherein the biosynthetic recombinant collagen has a single chain structure with fixed molecular weight in the range of 10 KDa to 100 KDa.

35. The method of claim 33, wherein, having a fixed molecular weight of 55 KDa.

36. The method of claim 34, wherein the single chain structure is in the form of a modified G-X-Y characteristic sequence which contains no immunogenic end.

37. The method of claim 1 , wherein the solution in the tube is vortexed for a period in the range of 3 minutes to 5 minutes.

38. The method of claim 37, wherein the solution is vortexed for a period of 3 minutes.

39. The method of claim 1 , wherein the solution in the container is placed in a shaker for a period in the range of 8 hours to 10 hours.

40. The method of claim 1 , wherein the volume of the sodium citrate collagen solution in the container is placed in a shaker for a period of 8 hours, at room temperature until a homogenous mixture is obtained.

41. The method of claim 1 , wherein the container with the volume of the sodium citrate collagen solution is placed in an oven with a temperature in the range of 55 °C to 65 °C.

42. The method of claim 41 , wherein the container with the volume of the sodium citrate collagen solution is placed in an oven with a temperature of 65 °C

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43. The method of claims 41 , wherein the container is placed in the oven for a period in the range of 8 hours to 12 hours.

44. The method of claim 43, wherein the container is placed in the oven for 12 hours.

45. The method of claim 1 , wherein a volume of 0.2ml up to 1.0 ml of a Sodium Chloride solution having a concentration rage of 0.5% up to 30% is added.

46. The method of claim 45, wherein 0.5ml Sodium Chloride of a 0.9% concentration is added.

47. The method of claim 1 , wherein the method is a single continuous method, within a single container.

48. A method of activating platelets and/or extracting growth factors from the platelets in blood, said method including:

Introducing a container having the solution of claim 1 ;

Adding blood to the container;

Centrifuging the container

Extracting the processed or part of the processed blood.

49. The method of claim 48, wherein extracting the processed or part of the processed blood, is the extraction of growth factors.

50. An extracted and processed blood, obtained from the method of claim 48, for use in regenerative medicine treatment.

51. An extracted product, obtained from the method of claim 48, for use in growth factor or cytokine rich regenerative medicine treatment.

52. The blood of claim 48, wherein the blood is human blood.

53. The blood of claim 48, wherein the blood is animal blood

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54. The blood of claim 48, wherein the blood is any other vertebrates blood.

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