Note: Descriptions are shown in the official language in which they were submitted.
CA 02814180 2016-12-09
A Method of Preserving Growth Factors Derived from Platelets
Cross-reference to Related Applications
This application claims the benefit of Hong Kong Short-term Patent
Application No. HK 12104129.2 filed on April 26, 2012.
Field of Invention
The present invention relates to a method of preserving growth factors
derived from platelets.
Background of Invention
Growth factors are naturally occurring proteins that are capable of
stimulating and enhancing cell growth and tissue repair. Growth factors are
stored and released in high concentration from activated platelet of fresh
blood.
Platelet rich plasma (PRP) is used for prolotherapy and skin rejuvenation
by injection to affected areas. However, PRP extracted from patient must be
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used fresh, and cannot be stored for a long period of time. The average life
span of platelets is typically 5 to 9 days after extraction.
A normal PRP treatment procedure requires a number of PRP treatments
for a period of 3 to 6 months. Typically, every time before receiving PRP
treatment, patients have to suffer venipuncture and wait for 30 minutes or
more
for the PRP to be extracted.
Therefore, it is vital to extend the shelf life of the growth factors in PRP
to
avoid multiple blood draws throughout the course of treatments, which saves
time and labor for both the physician and the patient from multiple
venipuncture and PRP extraction, and reduces the costs in processing the
PRP.
It is an objective of the present invention to provide an improved method
for preserving growth factors for an extended period of time capable of
spanning the whole treatment period.
Summary of invention
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,
The present method of preserving growth factors derived from platelets
includes the steps of:
a. obtaining a blood sample including platelets with growth factors
contained therein to be preserved;
b. releasing the growth factors from the platelets;
c. filtering out the released growth factors;
d. preserving the filtered growth factors;
whereby the growth factors are preserved for extended period of time.
Preferably, the growth factors are released by freezing and thawing the
platelets.
In an embodiment, the platelets are frozen at temperature between -50 C
and -60 C degree.
In another embodiment, the platelets are thawed at a temperature of 37 C
degree.
In another embodiment, the growth factors are lyophilized at temperature
between -40 C and -60 C.
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Preferably the preserving method includes the following major steps:
1. Obtaining a blood sample including platelets with growth factors
contained therein to be preserved.
2. Providing platelet rich plasma from the blood sample, for example, by
centrifugation.
3. Forming a mixture from the blood sample for stabilizing the pH value
therein.
4. Releasing the growth factors from the platelets, for example, by
freezing the platelets at temperature between -50 C degree and -60
C degree for 30 minutes and then thawing the platelets at a
temperature of 37 C degree for 10 minutes, and repeating the above
freezing and thawing for two more times.
5. Filtering out the released growth factors for preservation, for example,
by cellulose acetate membrane.
6. Freezing the filtered growth factors at a temperature of -55 C +/- 5 C
for at least 3 hours to prepare for lyophilization.
7. Lyophilizing the filtered growth factors at temperature between -40 C
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and -60 C and pressure of 0.18 millibar.
8. Preserving the growth factors at a temperature ranging from 2 C to
100c.
The present preservation method provides a stabilized form of the
lyophilized growth factor concentrate, which can be stored for a prolonged
period beyond 6 months, and is sufficient for the PRP treatment which
typically
lasts for 3 to 6 months. The resulting growth factors do not require special
storage means while the same can be preserved with high efficacy.
Further, the present preservation method eliminates multiple blood
extractions throughout the treatment period and the multiple processing of the
blood plasma for obtaining fresh growth factors. The preserved growth
factors are ready to be directly administered on the treatment areas.
These and other embodiments of the present invention are described in
greater details in the following detailed description of the invention. These
and
other objects, features, and advantages of the present invention will become
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,
apparent after the review of the following detailed description of the
disclosed
embodiments and claims.
Detail Description of the Invention
Collection of PRP
The first step of the present method is the extraction of patient's blood
sample including platelets with growth factors contained therein to be
preserved for future treatments. Typically, blood sample is to be extracted
from
the patient by means of venipuncture. It is preferably to obtain 30m1 to 60m1
of blood sample and store it in a sodium citrate blood collection tube for
ease
of transport.
The tube will be placed in a centrifuge at, for example, 3000 revolutions
per minute for the purpose of separating the PRP layer from blood cell layer.
Any other means for separating blood plasma from fresh blood may be
employed. The growth factors are now provided in a more concentrated form
in the platelet rich portion of centrifuged fresh blood.
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A buffering agent, for example, 0.8%
HEPES
(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) by weight is then added
to the PRP volume as a lyoprotectant. The addition of the buffering agent
stabilizes the pH value of PRP to avoid undesired fluctuations of pH value in
temperature extremities and maintains the PH value within a desired range, so
that the efficacy of growth factors will not be affected in the following
steps
including temperature change.
Decapsulation of Growth Factors
The next step is to release (decapsulate) the growth factors from the
platelets. To obtain a decapsulated growth factor solution, the extracted PRP
mixture may be frozen at temperature of -50 C to -60 C for 30 minutes, and
then thawed by placing the tube in water bath of 37 C for 10 minutes. By
way of freezing and thawing the mixture described herein, ice crystal
formations in the platelet expand in volume and eventually rupture the
platelet
membrane. Consequently, growth factors are released from the platelets and
a decapsulated growth factor solution is obtained. The freezing/thawing of the
solution is to be repeated for two more times with the aim of decapsulating as
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many growth factors as possible.
Platelets are prone to apoptosis at low temperature which may release
inhibitors and destroy the growth factors therein. However, decapsulated
growth factors which are released from platelet tend to be less vulnerable to
inhibitors. By releasing the growth factors from platelets, the preservation
period of growth factors can be much extended. The decapsulation of growth
factors from platelet (lysing of platelets) described in this invention can be
achieved in a number of ways including but not limited to freezing/thawing,
mechanical lysis, liquid homogenization, sonication and manual grinding.
Filtration
Subsequently, the solution undergoes a filtration process using, for
instance, a cellulose acetate membrane of 0.2 pm as filter. The purpose of
filtering the decapsulated growth factor solution is to reduce amount of
pathogens and other contaminants such as bacteria, red blood cells or cell
fragments which may affect the sterility of the solution and the life span of
the
growth factors.
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The solution is then distributed into serum vials and sealed with rubber
stoppers. These vials are to be frozen by a non-limited example at a
temperature of -55 C +/- 5 C for at least 3 hours to prepare for
lyophilization.
Lvophilization
Lyophilization, also known as freeze-drying, is a process which extracts
the water from the product by sublimation so that the product remains stable
and is easier to be stored at low temperature or room temperature.
To begin with lyophilization, the vials containing the released growth factor
solution, are retrieved from storage of temperature of, for example, -55 C.
The vials are left partially open to allow circulation of air and moisture to
escape during freeze-drying. The vials are then put into a freeze-dryer. The
freeze-drying process is maintained at temperature, by a non-limited example,
between -40 C and -60 C and pressure of about 0.18 millibar, for at least
ten
hours until samples of growth factors are dried thoroughly. Accordingly, a
powder of growth factor concentrate is formed (also known as cryo-crystalized
growth factor, C-GF).
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,
,
Upon visual inspection of the vials for sufficient dryness of the powder, the
vials are resealed, sealed air-tight and removed from the freeze-dryer to a
temperature, by a non-limited example, of 2 C - 10 C with humidity of lower
than 70%, which can be stored up to six months. The resulting growth factors
may then have shelf life as long as 22 months.
Administering the end product
The preserved growth factors are now readily be used by physician or
medical staff by rehydrating with 1:1 normal saline and applied as a serum on
areas of treatment. The preserved growth factors may also be administered
in form of powder directly on open moist wounds or ulcers, which is found to
be
an efficient way of treatment on patients.
The preserved growth factors can be stored for a prolonged period
beyond 6 months, wherein the time span of a PRP treatment is typically 3 - 6
months. The resulting growth factors may be stored in a typical household
refrigerator while the growth factors are preserved with high efficacy.
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The present invention is applicable on different kinds of growth factors.
Examples of growth factors which are beneficial by the present invention
include and are not limited to Platelet Derived Growth Factor (PDGF),
Transforming Growth Factor Beta (TGF-B), Insulin-like Growth Factor (1GF-1),
Platelet Factor-4 (PF-4), Vascular Endothelial Growth Factor (VEGF),
Epidermal Growth Factor (EGF), Hepatocyte Growth Factor (HGF), Bone
Morphogenetic Proteins (BMPs) and Fibroblast Growth Factor (FGF).
The foregoing description is given merely for clarity of understanding, and
no unnecessary limitations should be understood therefrom.
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