U.S. patent application number 11/719392 was filed with the patent office on 2009-03-19 for human serum for cell culture.
Invention is credited to Koji Suzuki, Seishin Tanaka.
Application Number | 20090075355 11/719392 |
Document ID | / |
Family ID | 36406993 |
Filed Date | 2009-03-19 |
United States Patent
Application |
20090075355 |
Kind Code |
A1 |
Suzuki; Koji ; et
al. |
March 19, 2009 |
HUMAN SERUM FOR CELL CULTURE
Abstract
It is intended to provide a serum which contains a large amount
of growth factors capable of efficiently promoting the growth of
stem cells. A human serum for cell culture which shows a residual
ratio of platelets remaining within 20 minutes after blood
collection in relation to the whole amount of the platelets is 0%
to 20%, and a release ratio of cell growth factors is 20% to
100%.
Inventors: |
Suzuki; Koji; (Hiroshima,
JP) ; Tanaka; Seishin; (Hiroshima, JP) |
Correspondence
Address: |
INTERNATIONAL KNOWLEDGE ASSET OFFICE
20 DANBURY LN.
IRVINE
CA
92618
US
|
Family ID: |
36406993 |
Appl. No.: |
11/719392 |
Filed: |
November 2, 2005 |
PCT Filed: |
November 2, 2005 |
PCT NO: |
PCT/JP05/20238 |
371 Date: |
May 15, 2007 |
Current U.S.
Class: |
435/183 |
Current CPC
Class: |
C12N 5/0018 20130101;
C12N 2501/15 20130101; A61K 35/16 20130101; C12N 2501/135
20130101 |
Class at
Publication: |
435/183 |
International
Class: |
C12N 9/00 20060101
C12N009/00 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 19, 2004 |
JP |
2004-335344 |
Claims
1. A human serum for cell culture obtained from a fluid comprising
humoral components derived from blood having blood coagulation
factors and platelets, and wherein a residual ratio of the
platelets remaining within 20 minutes after blood collection in
relation to the whole amount of the platelets is 0% to 20%, and a
release ratio of cell growth factors is 20% to 100%.
2. A human serum for cell culture obtained from a fluid comprising
humoral components derived from blood having blood coagulation
factors and platelets, and wherein a content of the cell growth
factors is greater than that of a human serum prepared from
plasma.
3. A human serum for cell culture obtained from a fluid comprising
humoral components derived from blood having blood coagulation
factors and platelets, and wherein a content of the cell growth
factors is greater than that of a human serum prepared by allowing
the blood to coagulate.
4. The human serum for cell culture according to claim 1, wherein
the cell growth factors comprise at least one of PDGF-BB and
TGF-.beta.f1.
5. The human serum for cell culture according to claim 1, wherein
the cell growth factors are obtained by bringing the fluid into
contact with a processed glass body.
6. The human serum for cell culture according to claim 5, wherein
the processed glass body is comprised of glass beads.
7. The human serum for cell culture according to claim 1, wherein
the human serum is prepared without being exposed to the
atmosphere.
8. The human serum for cell culture according to claim 1, wherein
the human serum is usable for a regenerative medicine method.
Description
TECHNICAL FIELD
[0001] The present invention relates to a human serum for cell
culture, and more particularly to a human serum for cell culture
including a large amount of cell growth factors.
[0002] This application is based upon and claims priority from
Japanese Patent Application No. 2004-335344, filed on Nov. 19,
2004, the entire contents of which are incorporated herein by
reference.
BACKGROUND ART
[0003] Currently, in the field of regenerative medicine, studies in
which stem cells collected from a subject are caused to proliferate
or differentiate ex vivo, and are thereafter transplanted into a
subject, thereby promoting regeneration of tissue of the subject,
have been carried out. Stem cells are multipotent and can
differentiate into a variety of tissues and organs, and they have
been attracting attention as cells which are the key to
regenerative medicine.
[0004] It has been known that in ex vivo cultural proliferation of
stem cells, the addition of a serum to the medium is effective.
However, when human therapies are targeted, the use of a serum
derived from an animal other than humans should be avoided in light
of possible safety problems. Therefore, the use of a serum prepared
from blood collected from a human, in particular, collected from
the same subject is desired. In addition, a culture of stem cells
in the field of regenerative medicine requires relatively larger
amounts of serum in comparison with blood tests.
[0005] As a method of preparing such a serum, a method in which a
blood collection tube is used that contains a blood coagulation
accelerating solid, such as glass powder, is disclosed (see Patent
Document 1). Furthermore, a method of facilitating collection of a
serum having a large amount and many kinds of growth factors by
bringing the blood into contact with glass powder so as to rapidly
separate a coagulation substance such as fibrin mixed in the serum
is disclosed (see Patent Document 2). Moreover, a method of
producing a serum as a raw material with human plasma is disclosed
(see Patent Document 3). In addition, a method of obtaining growth
factors by adding a calcium compound and glass beads to plasma is
disclosed (see patent document 4).
Patent Document 1: Japanese Unexamined Patent Application
Publication No. 2000-000228
[0006] Patent Document 2: Japanese Unexamined Patent Application
Publication No. Hei 04-83165
Patent Document 3: Japanese Unexamined Patent Application
Publication No. 2001-275662
Patent Document 4: Japanese Unexamined Patent Application
Publication No. 2004-269409
DISCLOSURE OF THE INVENTION
Problems to be Solved by the Invention
[0007] The method disclosed in patent document 1 or 2, however,
uses a low capacity blood collection tube designed for blood tests,
and therefore, preparatory procedures must be repeated many times
to prepare a serum in an amount required for a culture of stem
cells. Hence, this method is not suited for practical applications.
Furthermore, the method disclosed in patent document 3 uses
thrombin as an anticoagulant, and therefore, is not desirable
because it entails the infection risk caused by a substance of
biologic origin. Moreover, the method disclosed in patent document
4 cannot efficiently culture stem cells because it uses plasma
having only a small amount of cell growth factors as a raw
material, and accordingly, the resulting serum does not contain
sufficient cell growth factors.
[0008] The present invention was made in view of the foregoing
problems, and it is an object of the present invention to provide a
serum which contains a large amount of a growth factor capable of
efficiently promoting the growth of stem cells.
Means for Solving the Problems
[0009] More specifically, the following is provided.
[0010] In a first aspect of the present invention, a human serum
for cell culture obtained from a fluid comprising humoral
components derived from blood having blood coagulation factors and
platelets, and wherein a residual ratio of the platelets remaining
within 20 minutes after blood collection in relation to the whole
amount of the platelets is 0% to 20%, and a release ratio of cell
growth factors is 20% to 100%.
[0011] According to the first aspect of the present invention, the
release of the cell growth factors can be quickly promoted,
resulting from the residual ratio of the platelets remaining within
20 minutes after collection of the blood in relation to the whole
amount of the platelets is 0% to 20%. Furthermore, stem cells can
be efficiently proliferated, resulting from the release ratio of
cell growth factors being 20% to 100%. In addition, since the fluid
including at least humoral components and platelets derived from
blood is used, a serum having a larger amount of growth factors can
be produced in comparison with the case where the serum is prepared
from the plasma, thereby enabling the production of a serum having
a cell growth effect equivalent to a fetal bovine serum.
[0012] The term "blood" used herein indicates whole blood including
hemocytes (erythrocytes, leucocytes, platelets) and plasma (serum)
as a liquid component, and a liquid containing at least one of
these (for example, blood collected by apheresis). Furthermore, the
term "serum" used herein means a pale yellow liquid obtained by
allowing collected blood to stand, resulting in a reduction in
fluidity, followed by separation from the red coagulated block
(clot). The meaning of "serum" according to the present invention
is different from common serums in terms of the production process
not including separation from the clot, but it means a humoral
component in the blood that is useful in cell culture and that
includes coagulation factors and growth factors substantially
equivalent to those in common serums. The term "humoral component
derived from blood" used herein indicates "blood components other
than hemocytes" or "mixture of blood components other than
hemocytes and an agent such as an anticoagulant added thereto". The
term "cell growth factor" used herein means a platelet-derived
growth factor (PDGF), a transforming growth factor (TGF.beta.1), a
vascular endothelial growth factor (VEGF), an insulin-like growth
factor (IGF), a hepatocellular growth factor (HGF), a brain-derived
neurotrophic factor (BDNF), a basic fibroblast growth factor(bFGF),
or the like.
[0013] In addition, the term "release ratio of cell growth factors"
used herein indicates a ratio of an amount of cell growth factors
to a potential amount, where the amount of cell growth factors
contained in a serum prepared from a predetermined amount of blood
collected in a vacuum collection tube is assumed to be the
potential amount (100% of cell growth factors are released).
[0014] In a second aspect of the present invention, a human serum
for cell culture obtained from a fluid comprising humoral
components derived from blood having blood coagulation factors and
platelets, and wherein a content of the cell growth factors is
greater than that of a human serum prepared from plasma.
[0015] According to the second aspect of the present invention,
stem cells can be efficiently proliferated, due to the content of
the cell growth factors of the human serum being greater than that
of a human serum which is prepared from plasma. The term "plasma"
used herein indicates a supernatant liquid obtained by adding an
anticoagulant, such as heparin, CPD, or the like, to the collected
blood, followed by centrifugal separation. The term "human serum
prepared from plasma" used herein indicates a human serum obtained
by centrifuging blood collected from the subject under conditions
where the platelets are completely precipitated (for example, 4,400
(g).times.5 (min.) or greater) to prepare plasma, followed by
separating coagulation factors from the plasma.
[0016] In a third aspect of the present invention, a human serum
for cell culture obtained from a fluid comprising humoral
components derived from blood having blood coagulation factors and
platelets, and wherein a content of the cell growth factors is
greater than that of a human serum prepared by allowing the blood
to coagulate.
[0017] According to the third aspect of the present invention, stem
cells can be efficiently proliferated, due to the content of the
cell growth factors of the human serum being greater than that of a
human serum prepared by allowing the blood to coagulate. The term
"human serum prepared by allowing the blood to coagulate" used
herein indicates letting human serum obtained from blood collected
from the subject coagulate in a flexible vessel at room temperature
for approximately one hour, followed by centrifugal separation.
[0018] In a fourth aspect of the present invention, the human serum
for cell culture according to any one of the first to third
aspects, the cell growth factors comprise at least one of PDGF-BB
and TGF-.beta.1.
[0019] According to the fourth aspect of the present invention,
stem cells can be efficiently proliferated, due to the cell growth
factors comprising at least one of PDGF-BB and TGF-.beta.1, which
are higher among the growth factors in terms of cellular
proliferative potential. Here, PDGF-BB indicates one of three types
of PDGF (dimer) (PDGF-AA,PDGF-BB,PDGF-AB).
[0020] In a fifth aspect of the present invention, the human serum
for cell culture according to any one of the first to fourth
aspects, the cell growth factors are obtained by bringing the fluid
into contact with a processed glass body.
[0021] In order to activate the platelets in the fluid to have the
cell growth factors released, the fluid is required to be brought
into contact with foreign matter. In the human serum for cell
culture according to the fifth aspect of the present invention,
cell growth factors can be more efficiently released in comparison
with the case of a polyethylene pellet, as a result of the
processed glass body being used as the foreign matter. The
"processed glass body" used herein indicates glass powder, glass
beads, or the like. In order to reduce damage to erythrocytes
(hemolysis) and breakage of devices used to prepare the serum
according to the present invention, the shape of the processed
glass body is preferably formed to be nearly spherical.
[0022] Furthermore, with the aim of rapid activation of factors to
be activated such as platelets and coagulation factors, the surface
of the processed glass body is preferably formed with a layer
comprising a silicon dioxide compound. Examples of the silicon
dioxide compound which may be used include at least one selected
from glass, silica, diatomaceous earth, kaolin and the like, but
are not limited thereto.
[0023] In a sixth aspect of the present invention, the human serum
for cell culture according to the fifth aspect, the processed glass
body is comprised of glass beads.
[0024] According to the sixth aspect of the present invention
describes, the platelets in the fluid can be further activated, due
the processed glass body being comprised of glass beads. Among
them, porous glass beads are more preferable because they have a
large contact area with the fluid. Furthermore, the surface area of
the glass beads per 1 ml of the fluid is preferably in the range of
0.1 to 25 (mm.sup.2/ml).
[0025] In a seventh aspect of the present invention, the human
serum for cell culture according to any one of the first to sixth
aspects, the human serum is prepared without being exposed to the
atmosphere.
[0026] According to the seventh aspect of the present invention,
the risk of contamination by bacteria, microorganisms, or the like
can be reduced, resulting from the human serum being prepared
without being exposed to the atmosphere. Accordingly, a serum of
high safety can be produced in large quantities, thereby ensuring
high safety. Furthermore, the human serum for cell culture
according to the present invention is preferably prepared by such
an apparatus, which will prevent it from being exposed to the
atmosphere.
[0027] In an eighth aspect of the present invention, the human
serum for cell culture according to any one of the first to seventh
aspects, the human serum is usable for a regenerative medicine
method.
[0028] According to the eighth aspect of the present invention, use
of the serum as a medium for cell culture upon culturing by
inoculating stem cells collected from a subject to this medium can
culture the cells faster. Furthermore, because of the fact that the
cells can be cultured using an autologous serum, the probability of
adverse reaction or the like will be reduced, thereby being
superior in safety.
Effects of the Invention
[0029] As can be appreciated from the foregoing description, it is
to be understood that the human serum for cell culture according to
the present invention contains a large amount of a growth factor,
and accordingly, can more efficiently culture stem cells in
comparison with a conventional serum. Accordingly, the use of this
human serum for regenerative medicine ensures that tissues and
functions of the subject can be regenerated safely and
certainly.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] FIG. 1 illustrates a view of a serum preparation apparatus
for preparing a human serum for cell culture according to the
present invention;
[0031] FIG. 2 illustrates a view of a procedure of preparing a
human serum for cell culture according to the present
invention;
[0032] FIG. 3 illustrates a view of a state of shaking a blood
reservoir 10 of the serum preparation apparatus according to the
present invention;
[0033] FIG. 4 illustrates a graph of a relationship between elapsed
time following the shaking and the residual ratio of platelets in
each specimen;
[0034] FIG. 5 illustrates a graph of a relationship between elapsed
time following the shaking and the release ratio of PDGF-BB;
and
[0035] FIG. 6 illustrates a graph of a relationship between elapsed
time following the shaking and release ratio of TGF-.beta.1.
PREFERRED MODE FOR CARRYING OUT THE INVENTION
[0036] The present invention is described in detail below.
[0037] A human serum for cell culture according to the present
invention is preferably prepared with a serum preparation apparatus
for a cell culture for preparing a serum for a cell culturing
method that includes a serum preparing step of preparing a serum
containing a cell growth factor and a culturing step of culturing
cells in the presence of the prepared serum.
[0038] It is preferable that the serum preparation apparatus is
usable for cell culture, and includes a blood reservoir for holding
a fluid including at least humoral components and platelets derived
from the blood having blood coagulation factors, and the blood
reservoir has a serum producing function which produces a serum
suitable for a cell culturing step. It is further preferable that
the serum preparation apparatus is an apparatus of a closed system,
which can produce a serum without being exposed thereof to the
atmosphere. Here, the serum producing function is intended to
indicate a function of activating the platelets in the fluids, and
accordingly, increasing the content of the growth factors in a
serum so that the recovery of the serum is facilitated. This
function is provided by a blood coagulation accelerating solid
whose specific gravity is greater than that of the fluids.
[0039] More specifically, it is preferable that the serum is
prepared with the serum preparation apparatus described below, but
this is not limited thereto.
Serum Preparation Apparatus
[0040] As shown in FIG. 1, the serum preparation apparatus 1
comprises a blood reservoir 10, and a component storage member 20
as main elements. Among these, the blood reservoir 10 and the
component storage member 20 are constituted from a main body part
11 formed with two sheets of a flexible resin material, for
example, soft polyvinylchloride, fused to each other at the
external marginal part 11a to give a bag shape, and a processed
glass body 12 disposed inside of the main body part 11.
[0041] The glass processed bodies 12, serving as a blood
coagulation accelerating solid in the main body part 11, is
freely-movable in the main body part 11, each of the glass
processed bodies 12 having a substantially spherical shape composed
of, for example, soda glass. It is preferred to define the surface
area of the processed glass body 12 to satisfy a relationship to
the volume of reservable blood to be at least 0.1 mm.sup.2/ml, so
that activation of platelets and coagulation factors in the blood
is promoted.
[0042] In connection with the blood coagulation accelerating solids
in the blood reservoir, both a suspension of hemolysis and
activation promotion of platelets and coagulation factors during
the activation promoting step, and the centrifugal separation step
are made possible when the surface area of the processed glass body
12 is defined to satisfy the relationship of 0.1 mm.sup.2/ml to 25
mm.sup.2/ml in the volume of the blood which can be reserved in the
blood reservoir.
[0043] Two tubes 41 and 42 are connected in an air-tight manner at
the upper edge end of the main body part 11 of the blood reservoir
10 to the connection ports thereof, respectively. The tube 41 among
them serves as an introducing path for introducing the blood, and
accordingly, a needle for collecting blood 30 or a junction, which
can be connected to a needle for collecting blood, is connected at
the other end thereof. The serum preparation apparatus thus
constructed enables for the serum to be prepared from the collected
blood without atmospheric exposure.
[0044] The other tube 42 connected in an air-tight manner to the
blood reservoir 10 is connected to each of the bags 21 to 26 via
tubes 43 to 46 and 51 to 56, and Branches 61 to 65. These serve as
a discharging path for discharging separated blood components.
These tubes 41 to 46 and 51 to 56 are constituted from a resin
material having flexibility, for example, a material such as soft
polyvinylchloride or the like. In this configuration, the bags 21
to 26 and each tube 51 to 56 of the component storage member 20 are
also connected in an air-tight manner by, for example, solvent
adhesion, thermal welding, ultrasound welding or the like.
Serum Preparation Step
[0045] The serum preparation step using the serum preparation
apparatus 1 having the constitution as described above is explained
with reference to FIG. 2 and FIG. 3.
[0046] As shown in FIG. 2, the blood separation process using the
aforementioned serum preparation apparatus 1 is includes seven
steps (S1 to S7) as generally classified.
[0047] First, in the first step of the process, the needle for
collecting blood 30 shown in FIG. 1 is inserted into a subject
(patient), and blood is collected. In this step, the blood
collected from the needle for collecting blood 30 is stored in the
blood reservoir 10, which is positioned lower, via the tube 41
(reservation step S1). Here, the channel of the tube 42 is closed
on the blood reservoir 10 side by way of a clamp or the like such
that the collected blood in the blood reservoir 10 does not flow
into the component storage member 20. The reservation step S1 is
terminated after the required amount has been collected, taking
into account the patient's physical condition upon collecting the
blood. The required amount referred to herein may be approximately
200 to 600 ml when the physical constitution and physical condition
of the patient are problem free.
[0048] Next, as shown in FIG. 2, after initiating the reservation
step S1, the blood reservoir 10 is shaken (activation promoting
step S2) while the reservation step S1 is being carried out. As
shown in FIG. 3, the blood reservoir 10 storing the collected blood
is gently agitated by a shaking apparatus 100 brought into contact
with the glass processed bodies 12 stored inside. Then, the
platelets and coagulation factors consisted in the blood are
coagulated on the surface of the processed glass body 12, and from
the platelets activated during the coagulation, growth factors
derived therefrom are released. (Also, this activation promoting
step carried out at a low temperature is effective in the
acceleration of platelet agglutination.) Following the reservation
step S1, the needle for collecting blood 30 is removed from the
subject of the blood collection, and then a part of the tube 41
connecting the needle for collecting blood 30 with the blood
reservoir 10 is cut and fused, sealing its cut edge (fusion cutting
step S3) at the same time.
[0049] On the other hand, the blood reservoir 10 separated from the
patient proceeds through the activation promoting step S2 together
with the component storage member 20, and each of the tubes 42 to
46 and 51 to 56 connecting therebetween, as well as branches 61 to
65 and the like. They are bundled to be compact, and are subjected
to a centrifuge separation (centrifugal separation step S4).
Conditions for centrifugal separation of the blood reservoir 10 may
be defined depending on the amount of the reserved blood and type
of the components to be separated; however, they may be defined to
be, for example, 2,250 g.times.10 min, at 4.degree. C. The tube 42
is then maintained in the state with the channel being closed by a
breakable partition wall or a clamp, similar to the case of the
reservation step S1.
[0050] In cases where an anticoagulant is added previous to blood
collection, the fusion cutting step S3 and the centrifugal
separation step S4 may be carried out prior to the activation
promoting step S2. In this instance, the centrifugal separation may
be conducted under the following conditions: [0051] centrifugal
separation of whole blood: 4,400 g.times.4 to 6 min, 2,250
g.times.10 min; and [0052] centrifugal separation of platelet-rich
plasma (PRP): 1,100 g.times.4 to 6 min.
[0053] Referring back to FIG. 2, the factors to be activated
including hemocyte components, which were activated in the
activation promoting step S2 through the centrifugal separation
step S4, form a block shape and are separated from the blood
(separation step S5). Furthermore, the serum 71 separated and
extracted in the blood reservoir 10 in the separation step S5 is
sequentially divided into all or some of the bags 21 to 26 by
compressing the blood reservoir 10 (discharging step S6).
[0054] After the bag 21 is filled with a required amount of the
serum, the tube 51 is cut and sealed (fusion cutting step S7). This
cutting and sealing may be performed using a method that is similar
to the cutting and sealing of the tube 42 prior to the
aforementioned centrifugal separation step S4. Moreover, the bag 21
having the serum contained therein is subjected to a storage
treatment such as frozen storage.
[0055] The discharging step S6 and fusion cutting step S7 are
carried out sequentially on each of the bags 21 to 26, and the
operation for serum preparation is stopped when the serum is
contained in all or some of the bags 21 to 26. Additionally, the
erythrocytes may be washed and diluted, as needed, with
physiological saline, or an anticoagulant such as CPD, or ACD-A
liquid, or a liquid for preserving blood such as MAP, and can be
stored as blood for transfusion.
EXAMPLES
[0056] Hereinafter, the present invention is explained in more
detail, but not in any way to limit the present invention to these
Examples.
Example 1
[0057] Fresh human blood 20 (ml) was added to a vessel in which
five glass beads (.phi.: 4 mm, 50 mm.sup.2) were stored, and shaken
by a Multi Shaker (MMS-300, manufactured by Tokyo Rikakikai Co.,
Ltd.). At 10, 20, 30 and 60 minutes after shaking was initiated, 1
(ml) of blood was collected in a sampling tube in which an
anticoagulant was stored, and the blood was centrifuged to isolate
a serum. In addition, CPD-added blood was collected from the same
subject to isolate plasma. Calcium chloride was added to the
isolated plasma so that fibrin was deposited at 37.degree. C. and a
serum was prepared. The growth factors (PDGF-BB, TGF-.beta.1) were
measured for both the serum prepared from the vessel in which glass
beads were stored, and the serum prepared from the CPD-added blood.
The results are illustrated in Table 1. The amounts of the growth
factors (TGF-.beta.1, PDGF-BB) were measured by a commercially
available test kit manufactured by R&D SYSTEMS, Inc. using a
microplate reader (Multiskan BICHROMATIC manufactured by
Labsystem).
TABLE-US-00001 TABLE 1 Differences in content of growth factors
depending upon origins of the serum Origin of Derived from Vessel
containing Derived Serum five Glass Beads from CPD- Elapsed Time 10
20 30 60 Added Following Shaking minutes minutes minutes minutes
Plasma PDGF-BB 40.5 753.7 1677.2 1677.2 <32.5 pg/mL pg/mL pg/mL
pg/mL pg/mL TGF-.beta.1 21.2 24.4 26.4 29.6 1.5 ng/mL ng/mL ng/mL
ng/mL ng/mL
[0058] As for PDGF-BB, in the serum prepared from the vessel in
which five glass beads were contained, the content of PDGF-BB
increased as time elapsed following the shaking, and 1677.2 (pg) of
PDGF-BB was released per 1 ml of the serum after 60 minutes
elapsed. In the serum prepared from the plasma of the same subject,
the amount of PDGF-BB then was less than 32.5 (pg/ml) of the
detection limit. As for TGF-.beta.1, in the serum prepared from the
vessel in which five glass beads were contained, 21.2 (ng) of
TGF-.beta.1 had already been released per 1 (ml) after 10 minutes
of shaking. The amount released thereafter gradually increased in a
time dependent manner, and 29.6 (ng) of TGF-.beta.1 was released
after 60 minutes. On the other hand, in the serum prepared from the
plasma of the same subject, the amount of TGF-.beta.1 was 1.5 (ng)
per 1 (ml) of the serum.
Example 2
[0059] The following three types of vessels for collecting blood
were prepared. The first type of vessel for collecting blood
(specimen 3) contained no glass beads therein, the second type of
vessel for collecting blood (specimen 1) contained five glass beads
therein, and the third type of vessel for collecting blood
(specimen 2) contained 20 polyethylene pellets therein. 20 ml of
fresh blood derived from the same subject was added to each of the
vessels, and was shaken by a Multi Shaker (MMS-300, manufactured by
Tokyo Rikakikai Co., Ltd.). At 10, 20, 30 and 60 minutes after
shaking was initiated, 1 (ml) of each blood was collected in a
sampling tube containing an anticoagulant, and the number of
platelets was counted using a hemocryte counting apparatus
(Multiparameter automated hematology analyzer K-4500 manufactured
by SYSMEX CORPORATION).
[0060] Furthermore, each of the specimens was centrifuged to
isolate a serum, and the growth factors (PDGF-BB, TGF-.beta.1) were
measured for the serum. The amounts of the growth factors
(TGF-.beta.1, PDGF-BB) were measured by way of a commercially
available test kit manufactured by R&D SYSTEMS, Inc. using a
microplate reader (Multiskan BICHROMATIC manufactured by
Labsystem).
[0061] Moreover, blood was collected from the same subject into a
commercially available vacuum blood collection tube (VENOJECT II
manufactured by TERUMO CORPORATION) designed for a clinical
laboratory, and the collected blood was allowed to stand at room
temperature for approximately one hour, followed by isolation of a
serum; the growth factors were measured in the same manner. The
number of platelets is represented by a residual ratio of the
number of platelets to the number of platelets immediately after
blood collection, which is assumed to be 100%. The amount of each
of the cell growth factors contained in a serum prepared from a
vacuum collection tube is assumed to be a potential amount of each
of the growth factors contained in the platelets. The amount of
each of the growth factors is represented by a release ratio of an
amount of each of the cell growth factors of a serum obtained in a
period of elapsed time following the shaking to the potential
amount of each of the cell growth factors.
Number of Platelets
[0062] FIG. 4 illustrates a graph of a relationship between elapsed
time following the shaking and the residual ratio of platelets in
each specimen. The residual ratio of platelets in specimen 1 was
reduced to approximately 2% within 20 minutes after shaking had
been initiated. The residual ratio of platelets in specimen 2, on
the other hand, was reduced within 30 minutes after shaking had
been initiated, slightly behind specimen 1. Furthermore, it has
been revealed that the residual ratio of platelets in specimen 3
was reduced to less than 2% in 60 minutes after shaking had been
initiated, although it was rapidly reduced immediately after
shaking had been initiated.
Release Ratio of PDGF-BB
[0063] FIG. 5 illustrates a graph of a relationship between elapsed
time following the shaking and the release ratio of PDGF-BB in each
of the specimens. It has been discovered that, as for specimen 1,
PDGF-BB was rapidly released in a time dependent manner, so that
90% of the potential amount in the serum was released within an
hour after the shaking. As for the other two specimens, less than
20% of the amount of PDGF-BB to the potential amount in the serum
was released, although the number of platelets had been
reduced.
Release Ratio of TGF.beta.1
[0064] FIG. 6 illustrates a graph of a relationship between elapsed
time following the shaking and the release ratio of TGF-.beta.1 in
each of the specimens. As for the serum in specimen 1, nearly 70%
of the amount of TGF.beta.1 to the potential amount in the serum
was released within ten minutes after the shaking. As for the other
two specimens, although it has been found that the amount of the
release was increased, the release ratio of the amount of
TGF.beta.1 to the potential amount in the serum had remained
approximately 40% within sixty minutes after the shaking. As can be
seen from the aforementioned results, it has been revealed that the
polyethylene pellet plays a role similar to the glass beads in
terms of eliminating the platelets in the blood, but cannot
activate the platelets.
* * * * *