U.S. patent application number 13/754336 was filed with the patent office on 2013-06-06 for method for controlling proliferation of cord blood hematopoietic stem cells and use thereof.
This patent application is currently assigned to JMS CO., LTD.. The applicant listed for this patent is JMS CO., LTD.. Invention is credited to Satoshi HIRAI, Kasumi OGATA.
Application Number | 20130143322 13/754336 |
Document ID | / |
Family ID | 42395545 |
Filed Date | 2013-06-06 |
United States Patent
Application |
20130143322 |
Kind Code |
A1 |
HIRAI; Satoshi ; et
al. |
June 6, 2013 |
METHOD FOR CONTROLLING PROLIFERATION OF CORD BLOOD HEMATOPOIETIC
STEM CELLS AND USE THEREOF
Abstract
The present invention provides a method for controlling the
proliferation and differentiation of cord blood-derived
hematopoietic stem cells with excellent safety when proliferating
them by culturing. The hematopoietic stem cells are inoculated into
a medium containing a sonicated liquid component of cord blood. The
proliferation and differentiation of the cord blood hematopoietic
stem cells can be inhibited in the presence of the sonicated liquid
component of cord blood. On the contrary, the proliferation of cord
blood hematopoietic stem cells can be accelerated by inoculating
the hematopoietic stem cells into a medium containing a
non-sonicated liquid component of cord blood. Thus, according to
the present invention, by using serum derived from cord blood, it
is possible to regulate the inhibition of the proliferation and
differentiation of cord blood hematopoietic stem cells and the
acceleration of the proliferation of the same as desired.
Inventors: |
HIRAI; Satoshi;
(Hiroshima-shi, JP) ; OGATA; Kasumi;
(Hiroshima-shi, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
JMS CO., LTD.; |
Hiroshima-shi |
|
JP |
|
|
Assignee: |
JMS CO., LTD.
Hiroshima-shi
JP
|
Family ID: |
42395545 |
Appl. No.: |
13/754336 |
Filed: |
January 30, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13146613 |
Jul 27, 2011 |
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PCT/JP2010/050795 |
Jan 22, 2010 |
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13754336 |
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Current U.S.
Class: |
435/377 ;
435/289.1 |
Current CPC
Class: |
A61P 35/02 20180101;
C12N 5/0647 20130101; C12N 2500/84 20130101; C12N 2501/26 20130101;
C12N 2501/23 20130101; A61P 7/00 20180101; A61K 35/51 20130101;
C12N 2501/145 20130101 |
Class at
Publication: |
435/377 ;
435/289.1 |
International
Class: |
C12N 5/0789 20060101
C12N005/0789 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 27, 2009 |
JP |
2009-015639 |
Jan 14, 2010 |
JP |
2010-006277 |
Claims
1. A method for controlling proliferation of cord blood
hematopoietic stem cells, the method comprising the step of: (X)
inhibiting proliferation and differentiation of hematopoietic stem
cells by culturing the hematopoietic stem cells in a medium
containing a sonicated liquid component of cord blood.
2. The control method according to claim 1, further comprising the
step of: (Y) accelerating proliferation of the hematopoietic stem
cells by culturing the hematopoietic stem cells in a medium
containing a non-sonicated liquid component of cord blood.
3. The control method according to claim 2, wherein, in the step
(X), proliferation and differentiation of the hematopoietic stem
cells are inhibited, and then, in the step (Y), the proliferation
of the hematopoietic stem cells inhibited in the step (X) is
accelerated.
4. The control method according to claim 2, wherein, in the step
(Y), proliferation of the hematopoietic stem cells is accelerated,
and then, in the step (X), proliferation and differentiation of the
hematopoietic stem cells proliferated in the step (Y) are
inhibited.
5. The control method according to claim 1, wherein the
hematopoietic stem cells and the liquid component are derived from
cord blood of the same individual.
6. The control method according to claim 5, wherein the individual
is a mammal.
7. The control method according to claim 6, wherein the mammal is a
human.
8. A method for producing cord blood hematopoietic stem cells, the
method comprising the step of: controlling proliferation of
hematopoietic stem cells by the control method according to claim
1.
9. The production method according to claim 8, wherein
proliferation and differentiation of the hematopoietic stem cells
are inhibited by culturing the hematopoietic stem cells in the
medium containing the sonicated liquid component of cord blood
until a desired time at which proliferation of the hematopoietic
stem cells is started.
10. The production method according to claim 8, wherein the
hematopoietic stem cells are proliferated, and then, proliferation
and differentiation of the proliferated hematopoietic stem cells
are inhibited by culturing the proliferated hematopoietic stem
cells in the medium containing the sonicated liquid component of
cord blood until a desired time at which the hematopoietic stem
cells are used.
11. The production method according to claim 8, wherein
proliferation of the hematopoietic stem cells is accelerated by
culturing the hematopoietic stem cells in a medium containing a
non-sonicated liquid component of cord blood.
12-19. (canceled)
20. A cord blood component preparation device for preparing cord
blood hematopoietic stem cells, a sonicated liquid component of
cord blood, and a non-sonicated liquid component of cord blood from
the same cord blood, for use in the method for controlling
proliferation of cord blood hematopoietic stem cells according to
claim 1, the cord blood component preparation device comprising: a
storage unit for storing cord blood; an erythrocyte accommodation
unit for accommodating erythrocytes; a hematopoietic stem cell
accommodation unit for accommodating hematopoietic stem cells;
first and second plasma accommodation units for accommodating
plasma; and first and second serum accommodation units for
accommodating serum, wherein the storage unit is connected to each
of the erythrocyte accommodation unit, the hematopoietic stem cell
accommodation unit, and the first and second plasma accommodation
units, the first plasma accommodation unit is connected to the
first serum accommodation unit, the second plasma accommodation
unit is connected to the second serum accommodation unit, the
storage unit is configured so that an erythrocyte sedimenting agent
can be introduced thereto, the erythrocyte accommodation unit is
configured so that erythrocytes having sedimented from cord blood
in the storage unit can be introduced thereto, the hematopoietic
stem cell accommodation unit is configured so that hematopoietic
stem cells having sedimented from a supernatant obtained after the
sedimentation of the erythrocytes in the storage unit can be
introduced thereto, the first plasma accommodation unit and the
second plasma accommodation unit are configured so that the
supernatant obtained after the sedimentation of the erythrocytes
and the supernatant obtained after the sedimentation of the
hematopoietic stem cells in the storage unit can be introduced
thereto, respectively, the first plasma accommodation unit is
configured so that sonication and a fibrin-forming treatment with
respect to the supernatant introduced thereto can be performed
therein, the first serum accommodation unit is configured so that,
among a sediment fraction and a supernatant fraction obtained from
the supernatant after being subjected to the sonication and the
fibrin-forming treatment in the first plasma accommodation unit,
the supernatant fraction can be introduced thereto as sonicated
serum, the second plasma accommodation unit is configured so that a
fibrin-forming treatment with respect to the supernatant introduced
thereto can be performed therein, and the second serum
accommodation unit is configured so that, among a sediment fraction
and a supernatant fraction obtained from the supernatant after
being subjected to the fibrin-forming treatment in the second
plasma accommodation unit, the supernatant fraction can be
introduced thereto as non-sonicated serum.
21. A cord blood component preparation device for preparing cord
blood hematopoietic stem cells, a sonicated liquid component of
cord blood, and a non-sonicated liquid component of cord blood from
the same cord blood, for use in the method for controlling
proliferation of cord blood hematopoietic stem cells according to
claim 1, the cord blood component preparation device comprising: a
storage unit for storing cord blood; a cell-containing plasma
accommodation unit for accommodating plasma containing
hematopoietic stem cells; a hematopoietic stem cell accommodation
unit for accommodating hematopoietic stem cells; first and second
plasma accommodation units for accommodating plasma; and first and
second serum accommodation units for accommodating serum, wherein
the storage unit is connected to the cell-containing plasma
accommodation unit, the cell-containing plasma accommodation unit
is connected to each of the hematopoietic stem cell accommodation
unit and the first and second plasma accommodation units, the first
plasma accommodation unit is connected to the first serum
accommodation unit, the second plasma accommodation unit is
connected to the second serum accommodation unit, the storage unit
is configured so that an erythrocyte sedimenting agent can be
introduced thereto, the cell-containing plasma accommodation unit
is configured so that, among a sediment fraction containing
erythrocytes having sedimented from cord blood in the storage unit
and a supernatant fraction, the supernatant fraction can be
introduced thereto as plasma containing hematopoietic stem cells,
the hematopoietic stem cell accommodation unit is configured so
that hematopoietic stem cells having sedimented from the plasma
containing hematopoietic stem cells in the cell-containing plasma
accommodation unit can be introduced thereto, the first plasma
accommodation unit and the second plasma accommodation unit are
configured so that a supernatant obtained after removing the
hematopoietic stem cells in the cell-containing plasma
accommodation unit can be introduced thereto, the first plasma
accommodation unit is configured so that sonication and a
fibrin-forming treatment with respect to the supernatant introduced
thereto can be performed therein, the first serum accommodation
unit is configured so that, among a sediment fraction and a
supernatant fraction obtained from the supernatant after being
subjected to the sonication and the fibrin-forming treatment in the
first plasma accommodation unit, the supernatant fraction can be
introduced thereto as sonicated serum, the second plasma
accommodation unit is configured so that a fibrin-forming treatment
with respect to the supernatant introduced thereto can be performed
therein, and the second serum accommodation unit is configured so
that, among a sediment fraction and a supernatant fraction obtained
from the supernatant after being subjected to the fibrin-forming
treatment in the second plasma accommodation unit, the supernatant
fraction can be introduced thereto as non-sonicated serum.
22. A cord blood component preparation device for preparing cord
blood hematopoietic stem cells, a sonicated liquid component of
cord blood, and a non-sonicated liquid component of cord blood from
the same cord blood, for use in the method for controlling
proliferation of cord blood hematopoietic stem cells according to
claim 1, the cord blood component preparation device comprising: a
storage unit for storing cord blood; a cell-containing plasma
accommodation unit for accommodating plasma containing
hematopoietic stem cells; first and second plasma accommodation
units for accommodating plasma; and first and second serum
accommodation units for accommodating serum, wherein the storage
unit is connected to the cell-containing plasma accommodation unit,
the cell-containing plasma accommodation unit is connected to each
of the first and second plasma accommodation units, the first
plasma accommodation unit is connected to the first serum
accommodation unit, the second plasma accommodation unit is
connected to the second serum accommodation unit, the storage unit
is configured so that an erythrocyte sedimenting agent can be
introduced thereto, the cell-containing plasma accommodation unit
is configured so that, among a sediment fraction containing
erythrocytes having sedimented from cord blood in the storage unit
and a supernatant fraction, the supernatant fraction can be
introduced thereto as a plasma fraction containing hematopoietic
stem cells, and after the plasma fraction introduced thereto is
separated into a sediment fraction containing the hematopoietic
stem cells and a supernatant fraction, the cell-containing plasma
accommodation unit can be divided into a lower region containing
the sediment fraction and an upper region containing the
supernatant fraction, the lower region of the cell-containing
plasma accommodation unit serves as a hematopoietic stem cell
accommodation unit, the first plasma accommodation unit and the
second plasma accommodation unit are configured so that the
supernatant fraction in the upper region of the cell-containing
plasma accommodation unit can be introduced thereto, the first
plasma accommodation unit is configured so that sonication and a
fibrin-forming treatment with respect to the supernatant introduced
thereto can be performed therein, the first serum accommodation
unit is configured so that, among a sediment fraction and a
supernatant fraction obtained from the supernatant after being
subjected to the sonication and the fibrin-forming treatment in the
first plasma accommodation unit, the supernatant fraction can be
introduced thereto as a sonicated serum fraction, the second plasma
accommodation unit is configured so that a fibrin-forming treatment
with respect to the supernatant introduced thereto can be performed
therein, and the second serum accommodation unit is configured so
that, among a sediment fraction and a supernatant fraction obtained
from the supernatant after being subjected to the fibrin-forming
treatment in the second plasma accommodation unit, the supernatant
fraction can be introduced thereto as non-sonicated serum.
23. A cord blood component preparation device for preparing cord
blood hematopoietic stem cells, a sonicated liquid component of
cord blood, and a non-sonicated liquid component of cord blood from
the same cord blood, for use in the method for controlling
proliferation of cord blood hematopoietic stem cells according to
claim 1, the cord blood component preparation device comprising:
first and second storage units for storing cord blood; an
erythrocyte accommodation unit for accommodating erythrocytes; a
hematopoietic stem cell accommodation unit for accommodating
hematopoietic stem cells; first and second plasma accommodation
units for accommodating plasma; and first, second, and third serum
accommodation units for accommodating serum, wherein the first
storage unit is connected to each of the erythrocyte accommodation
unit, the hematopoietic stem cell accommodation unit, and the first
and second plasma accommodation units, the first plasma
accommodation unit is connected to the first serum accommodation
unit, the second plasma accommodation unit is connected to the
second serum accommodation unit, the first storage unit is
configured so that an anticoagulant and an erythrocyte sedimenting
agent can be introduced thereto, the erythrocyte accommodation unit
is configured so that erythrocytes having sedimented from cord
blood in the storage unit can be introduced thereto, the
hematopoietic stem cell accommodation unit is configured so that
hematopoietic stem cells having sedimented from a supernatant
obtained after the sedimentation of the erythrocytes in the storage
unit can be introduced thereto, the first plasma accommodation unit
and the second plasma accommodation unit are configured so that the
supernatant obtained after the sedimentation of the erythrocytes
and the supernatant obtained after the sedimentation of the
hematopoietic stem cells in the storage unit can be introduced
thereto, respectively, the first plasma accommodation unit is
configured so that sonication and a fibrin-forming treatment with
respect to the supernatant introduced thereto can be performed
therein, the first serum accommodation unit is configured so that,
among a sediment fraction and a supernatant fraction obtained from
the supernatant after being subjected to the sonication and the
fibrin-forming treatment in the first plasma accommodation unit,
the supernatant fraction can be introduced thereto as sonicated
serum, the second plasma accommodation unit is configured so that a
fibrin-forming treatment with respect to the supernatant introduced
thereto can be performed therein, the second serum accommodation
unit is configured so that, among a sediment fraction and a
supernatant fraction obtained from the supernatant after being
subjected to the fibrin-forming treatment in the second plasma
accommodation unit, the supernatant fraction can be introduced
thereto as non-sonicated serum, the second storage unit is
configured so that a blood coagulation-accelerating substance for
accelerating blood coagulation can be introduced thereto, and the
third serum accommodation unit is configured so that a supernatant
fraction obtained after blood coagulation in the storage unit can
be introduced thereto.
Description
TECHNICAL FIELD
[0001] The present invention relates to a method for controlling
the proliferation of cord blood-derived hematopoietic stem cells
and the use thereof. More specifically, the present invention
further relates to a method for producing the hematopoietic stem
cells, and to a proliferation-controlling agent, a
proliferation-controlling kit, and a cord blood component
preparation device used in the control method.
BACKGROUND ART
[0002] Hematopoietic stem cells are multipotent stem cells having
self-renewal potential and capable of differentiating into all
types of hematopoietic cells such as leukocytes, erythrocytes, and
platelets. They are present in bone marrow fluid, peripheral blood,
and cord blood. It is known that hematopoietic stem cell
transplant, which is the transplant of hematopoietic stem cells
into a body, is an effective way of treating intractable blood
disease such as leukemia. Although hematopoietic stem cells in bone
marrow fluid or peripheral blood generally are used for the
treatment, there is a problem in that their collection places a
considerable burden on donors. In contrast, cord blood is obtained
secondarily at the time of delivery, so that it places a lower
burden on donors. Besides, cord blood is excellent in transplant
compatibility. Thus, cord blood is attracting attention as a supply
source of hematopoietic stem cells.
[0003] However, the amount of cord blood obtained from a donor is
smaller than the amount of bone marrow fluid or peripheral blood,
so that the cord blood may fail to provide a sufficient number of
cells necessary for transplant. As a remedy therefor, there has
been developed a method for proliferating cord blood-derived
hematopoietic stem cells by culturing so that they can be used for
transplant. As a specific example thereof, a method for
accelerating the proliferation of the hematopoietic stem cells by
culturing them together with mesenchymal stem cells, feeder cells
derived from an animal of a different species, or the like has been
reported (Patent Document 1 and Patent Document 2). When culturing
the hematopoietic stem cells, it is desired to inhibit the
proliferation and differentiation until a desired time, for
example. Thus, for example, a method in which a virus vector
incorporating a differentiation-inhibiting gene is transfected into
the hematopoietic stem cells has been reported (Patent Document 3).
However, regarding the method including the transfection of the
virus vector, there is a fear that it may affect the expression of
other genes, so that its safety might be insufficient, for example.
It also has a problem of cumbersome operations and high cost.
CITATION LIST
Patent Document
[0004] [Patent Document 1] JP 2006-61106 A [0005] [Patent Document
2] JP 2007-202506 A [0006] [Patent Document 3] JP 1999-127859 A
BRIEF SUMMARY OF THE INVENTION
Problem to be Solved by the Invention
[0007] With the foregoing in mind, it is an object of the present
invention to provide a method for controlling the proliferation and
differentiation of cord blood-derived hematopoietic stem cells with
excellent safety when proliferating them by culturing.
Means for Solving Problem
[0008] In order to achieve the above object, a control method
according to the present invention is a method for controlling
proliferation of cord blood hematopoietic stem cells, including the
step of:
(X) inhibiting proliferation and differentiation of hematopoietic
stem cells by culturing the hematopoietic stem cells in a medium
containing a sonicated liquid component of cord blood.
[0009] A production method according to the present invention is a
method for producing cord blood hematopoietic stem cells, including
the step of controlling proliferation of hematopoietic stem cells
by the control method according to the present invention.
[0010] A proliferation-controlling agent according to the present
invention is a proliferation-controlling agent for use in the
control method according to the present invention. The
proliferation-controlling agent contains:
(a) a proliferation inhibitor for inhibiting proliferation and
differentiation of the hematopoietic stem cells, the proliferation
inhibitor containing a sonicated liquid component of cord
blood.
[0011] A proliferation-controlling kit according to the present
invention is a proliferation-controlling kit for use in the control
method according to the present invention. The
proliferation-controlling kit includes the proliferation inhibitor
(a).
Effects of the Invention
[0012] According to the present invention, it is possible to
control the proliferation and differentiation of cord blood
hematopoietic stem cells using the liquid component derived from
cord blood without using a virus vector having a problem in safety
as described above, for example. Specifically, it is possible to
inhibit the proliferation and differentiation using only a
sonicated liquid component of cord blood. Therefore, the present
invention is very excellent in safety and can regulate the
proliferation and differentiation with simple operations. In
particular, since the present invention uses a liquid component of
cord blood, it is possible to use a liquid component derived from
cord blood of the same individual as the hematopoietic stem cells,
for example. Therefore, cord blood can be used more effectively,
and since the components derived from the same individual can be
used in combination, the reliability regarding the safety also can
be improved. As described above, since the present invention can
inhibit the proliferation and differentiation of cord blood
hematopoietic stem cells, it is particularly useful when delivering
the hematopoietic stem cells to a destination, storing the
hematopoietic stem cells until a desired time at which the
proliferation of the hematopoietic stem cells is started, and the
like, for example. Thus, it can be said that the method according
to the present invention can promote further the effective
utilization of cord blood hematopoietic stem cells in the field of
regenerative medicine.
BRIEF DESCRIPTION OF DRAWINGS
[0013] FIG. 1 is a plan view schematically showing an example of a
cord blood component preparation device according to the present
invention.
[0014] FIG. 2 is a plan view schematically showing an example of
another cord blood component preparation device according to the
present invention.
[0015] FIG. 3 is a plan view schematically showing an example of
still another cord blood component preparation device according to
the present invention.
[0016] FIG. 4 is a perspective view showing an example of a method
of using a cell-containing plasma accommodation unit in the cord
blood component preparation device according to the present
invention.
[0017] FIG. 5 is a plan view showing an example of another
cell-containing plasma accommodation unit in the cord blood
component preparation device according to the present
invention.
[0018] FIG. 6 is a plan view schematically showing an example of
still another cord blood component preparation device according to
the present invention.
[0019] FIG. 7 is a graph showing the number of hematopoietic stem
cells cultured in the presence of serum in Example 1.
MODE FOR CARRYING OUT THE INVENTION
[0020] <Proliferation Control Method>
[0021] The control method according to the present invention is, as
described above, a method for controlling proliferation of cord
blood hematopoietic stem cells, including the step of:
(X) inhibiting proliferation and differentiation of hematopoietic
stem cells by culturing the hematopoietic stem cells in a medium
containing a sonicated liquid component of cord blood.
[0022] In the present invention, the sonicated liquid component of
cord blood hereinafter also is referred to as the "treated liquid
component". In general, blood is roughly composed of a liquid
component (liquid fraction) and a cell component (cell fraction).
The liquid component is serum or plasma, for example. The cell
component is a blood cell component (blood cell fraction), which
includes erythrocytes, leukocytes, and platelets, for example. In
the present invention, the liquid component may be either serum or
plasma, for example. In the present invention, it is only necessary
that the medium contains the liquid component, and other
configurations are by no means limited. Although the liquid
component of cord blood contained in the medium further may
contain, for example, cord blood-derived components such as the
cell component and a blood coagulation factor, it is preferable
that the liquid component of cord blood contained in the medium is:
a liquid component obtained by removing the cell component from the
cord blood; a liquid component obtained by removing the blood
coagulation factor from the cord blood; or a liquid component
obtained by removing the cell component and the blood coagulation
factor from the cord blood. It is to be noted that the removal of
the cell component and the removal of the blood coagulation factor
are not limited to complete removal thereof from the cord blood.
The blood coagulation factor is fibrinogen (Factor I), prothrombin
(Factor II), or the like, for example.
[0023] In the present invention, the cord blood to be sonicated is
not particularly limited, and may be, for example, the cord blood
itself or a liquid component thereof. A liquid component before
sonication hereinafter also is referred to as a "not-yet-treated
liquid component". Examples of the liquid component include plasma
and serum. The "plasma" generally is a liquid fraction obtained by
removing the blood cells from blood, and the "serum" generally is a
liquid fraction obtained by removing the blood cells and some kinds
of blood coagulation factors from blood. In the case where the
liquid component is sonicated, the liquid component may be, for
example, any of a liquid fraction obtained by removing the blood
cell component from cord blood; a liquid fraction obtained by
removing the blood coagulation factor from cord blood; and a liquid
fraction obtained by removing the blood cell component and the
blood coagulation factor from cord blood. Among them, a liquid
fraction obtained by removing the blood cell component from cord
blood is preferable, for example. Furthermore, in the case where
the liquid component is sonicated, the liquid component may contain
platelets, for example. Examples of plasma containing the platelets
include so-called platelet-rich plasma.
[0024] In the present invention, the treated liquid component may
be prepared by sonicating cord blood itself and then collecting a
liquid component, or by collecting a liquid component from cord
blood and then sonicating the thus-collected liquid component, for
example. When collecting the liquid component from the cord blood,
it is preferable to remove not only the blood cells but also blood
coagulation factors such as fibrinogen, for example.
[0025] In the present invention, the liquid component (treated
liquid component) of cord blood preferably is serum (treated serum)
obtained from sonicated cord blood plasma, for example.
[0026] The present invention further may include the step of:
(Y) accelerating proliferation of the hematopoietic stem cells by
culturing the hematopoietic stem cells in a medium containing a
non-sonicated liquid component of cord blood.
[0027] The non-sonicated liquid component of cord blood hereinafter
is referred to as the "untreated liquid component". Similarly to
the above-described treated liquid component, examples of the
untreated liquid component also include plasma and serum.
[0028] In the present invention, either the step (X) of inhibiting
the proliferation and differentiation or the step (Y) of
accelerating the proliferation may be performed first, for example.
The method according to a first embodiment is such that, for
example, first, in the step (X), proliferation and differentiation
of the hematopoietic stem cells are inhibited using the treated
liquid component, and thereafter, in the step (Y), the
proliferation of the hematopoietic stem cells inhibited in the step
(X) is accelerated using the untreated liquid component. According
to this embodiment, it is possible to inhibit the proliferation and
differentiation of the hematopoietic stem cells until a desired
time so as to start the proliferation of the hematopoietic stem
cells at the desired time. That is, it is possible to control the
ON-OFF of the proliferation of the hematopoietic stem cells easily
merely by changing the serum to be used.
[0029] Furthermore, the method according to a second embodiment is
such that, for example, first, in the step (Y), the proliferation
of the hematopoietic stem cells is accelerated using the untreated
liquid component, and thereafter, in the step (X), proliferation
and differentiation of the hematopoietic stem cells proliferated in
the step (Y) are inhibited using the treated liquid component.
According to this embodiment, the proliferated hematopoietic stem
cells can be stored while preventing further proliferation and
differentiation thereof until a desired time.
[0030] In the present invention, hematopoietic stem cells to be
controlled are derived from cord blood, and serum used to control
the proliferation of the hematopoietic stem cells also is derived
from cord blood. Thus, in the present invention, it is possible to
use cord blood hematopoietic stem cells and a liquid component of
cord blood derived from the same individual, for example.
Specifically, the proliferation and differentiation can be
inhibited using hematopoietic stem cells and a treated liquid
component derived from cord blood of the same individual, and
further, the proliferation can be accelerated using an untreated
liquid component derived from the cord blood of the same
individual. Thus, among components of cord blood, not only
hematopoietic stem cells but also a liquid component can be used to
control the proliferation of the hematopoietic stem cells, so that
the cord blood can be utilized still more effectively.
[0031] The individual is not particularly limited, and examples
thereof include: humans; rodents; domestic animals; and mammals
such as primates, excluding humans.
[0032] In the present invention, the method for preparing
hematopoietic stem cells is not particularly limited, and any
conventionally known method can be employed. An example of the
method will be described below, but the method is not limited
thereto. First, erythrocytes in cord blood are caused to sediment,
and a supernatant fraction is collected. Cord blood is blood
contained in a placenta and an umbilical cord. It generally is
collected from a placenta and an umbilical cord during or after a
delivery and placed in a blood collection bag containing an
anticoagulant at a hospital. An erythrocyte sedimenting agent for
separating erythrocytes is added to the cord blood containing the
anticoagulant. Examples of the erythrocyte sedimenting agent
include HES (Hydroxy Ethyl Starch). The amount of the erythrocyte
sedimenting agent to be added is not particularly limited.
Preferably, the erythrocyte sedimenting agent is added to cord
blood so that its concentration becomes 1 to 50 mg/ml, for example.
By allowing the cord blood containing the erythrocyte sedimenting
agent to stand still, the cord blood separates into a fraction
containing erythrocytes and a supernatant fraction containing
hematopoietic stem cells. The conditions for carrying out this
separation are not particularly limited. For example, the
separation is carried out at a temperature from 15.degree. C. to
25.degree. C. For example, when the cord blood contains the
erythrocyte sedimenting agent in advance, it is not necessary to
add the erythrocyte sedimenting agent (the same applies
hereinafter).
[0033] Next, the supernatant fraction is separated into a sediment
fraction containing hematopoietic stem cells and a liquid fraction
(supernatant fraction) by centrifuging. The conditions for the
centrifuging are not particularly limited, and are as follows, for
example: the centrifugal acceleration is 10780 to 43120 m/s.sup.2
(1100 to 4400.times.g); the temperature is 2.degree. C. to
37.degree. C.; and the time is 4 to 30 minutes. In the present
invention, it is preferable to use hematopoietic stem cells and a
liquid fraction derived from the same individual, as described
above. Thus, the liquid fraction obtained after centrifuging
preferably is used for the preparation of the liquid component, as
will be described below. The liquid fraction obtained after the
centrifuging may be a fraction containing plasma (plasma fraction)
or a fraction containing serum (serum fraction), for example.
Furthermore, the liquid fraction obtained after centrifuging
further may contain, for example, platelets, as described above. As
described above, the liquid component may be prepared from a liquid
component containing platelets, for example. Thus, when collecting
the sediment fraction, centrifuging may be carried out under
conditions such that the liquid fraction obtained after
centrifuging contains platelets, for example. Such conditions are
not particularly limited, and centrifuging can be carried out
according to a conventionally known method for collecting
platelet-rich plasma, for example. Specifically, the conditions are
as follows, for example: the centrifugal acceleration is 2940 to
11760 m/s.sup.2 (300 to 1200.times.g), the temperature is 1.degree.
C. to 20.degree. C., and the time is 3 to 6 minutes.
[0034] Subsequently, hematopoietic stem cells are purified from the
sediment fraction. Specifically, first, magnetic beads having an
anti-CD34 antibody immobilized thereon are added to the sediment
fraction, thereby causing CD34 of the hematopoietic stem cells to
react with the anti-CD34 antibody on the magnetic beads. Through
this antigen-antibody reaction, the hematopoietic stem cells are
bound to the magnetic beads. Subsequently, the sediment fraction to
which the magnetic beads have been added is applied to a magnetic
column so as to trap the magnetic beads in the column by a magnetic
force, and unnecessary components in the sediment fraction are
removed. In this manner, purified hematopoietic stem cells can be
prepared.
[0035] In the present invention, the method for preparing a treated
liquid component derived from cord blood is not particularly
limited. In the present invention, it is preferable to use
hematopoietic stem cells and a liquid component derived from cord
blood of the same individual. Thus, as an illustrative example, a
method for carrying out the preparation of the liquid component
along with the above-described preparation of hematopoietic stem
cells will be described.
[0036] First, in the manner described above, cord blood is
separated into a sediment fraction containing hematopoietic stem
cells and a liquid fraction (supernatant fraction). The liquid
fraction may be, for example, a plasma fraction or a serum
fraction, as described above. Then, the liquid fraction is
sonicated. The method for carrying out the sonication is not
particularly limited, and examples thereof include the following
method. The conditions for the sonication are not particularly
limited. For example, the sonication is carried out under the
conditions where, when ultrasonic waves against an object to be
treated are measured with a sound pressure meter, a sound pressure
of at least 5 mV can be obtained, with the frequency being set to,
for example, 28 to 100 kHz, the distance from an ultrasonic
generator being set to, for example, 5 to 30 cm, and the treatment
time being set to, for example, 5 to 60 minutes, preferably 10 to
30 minutes. In the present invention, ultrasonic waves can be
generated using an ordinary ultrasonic generator, for example. The
treated liquid component can be obtained in this manner.
[0037] In the case where the liquid fraction contains a blood
coagulation factor such as fibrinogen, for example, the blood
coagulation factor further is removed from the liquid fraction, and
the liquid fraction after the removal can be used as the treated
liquid component, for example. Specifically, for example, when the
liquid fraction is a plasma fraction containing the blood
coagulation factor, it is preferable that the blood coagulation
factor is removed, serum is collected, and the thus-collected serum
is used as the treated liquid fraction. The removal of the blood
coagulation factor may be performed either before or after the
sonication, for example. The method for removing the blood
coagulation factor is not particularly limited, and examples
thereof include a method in which a blood coagulation factor such
as fibrinogen is denatured and then removed as an insoluble
fraction. A specific example thereof is a method in which
fibrinogen is denatured so as to form fibrin and the thus-formed
fibrin is removed as an insoluble fraction, for example. The
denaturing method is not particularly limited, and examples thereof
include thermal denaturation. Specifically, examples of the
denaturing method include heat-treating the liquid fraction. The
heating conditions are not particularly limited, and are as
follows, for example: the heating temperature preferably is
50.degree. C. to 60.degree. C.; and the heating time preferably is
10 to 120 minutes, more preferably 30 to 60 minutes. The insoluble
fraction can be removed by allowing the heat-treated liquid
fraction to stand still, centrifuging the heat-treated liquid
fraction, or filtering the heat-treated liquid fraction, for
example. Then, the treated liquid component is obtained by
collecting a supernatant fraction obtained after the standing
still, the supernatant fraction obtained after the centrifuging, or
a filtrate obtained after the filtration, for example. The
conditions for the standing still, centrifuging, filtering, or the
like are by no means limited, and conventionally known conditions
can be employed. Furthermore, examples of the method for removing
the blood coagulation factor from the liquid fraction include, in
addition to the above methods, methods utilizing the activation
mechanism of a blood coagulation system, e.g., adding a coagulation
factor such as calcium chloride or thrombin to the liquid fraction.
Specific conditions thereof are by no means limited, and
conventionally known conditions can be employed.
[0038] On the other hand, the untreated liquid component can be
prepared in the same manner as in the above, except that the
sonication is not performed, for example.
[0039] In the present invention, the step (X), i.e., the step of
inhibiting the proliferation and differentiation of hematopoietic
stem cells, can be carried out by inoculating the hematopoietic
stem cells into a medium containing the sonicated liquid component,
as described above. As a specific example thereof, an example where
the treated liquid component serum is treated serum will be
described below. It is to be noted, however, that the present
invention is not limited thereto, and treated liquid components
other than serum also can be used, for example.
[0040] It is only necessary that the medium contains the treated
serum, and other configurations and conditions are by no means
limited. Examples of the medium include media that can be used for
culturing hematopoietic stem cells of cord blood. As specific
examples, an Iscove's modified Dulbecco's medium (IMDM), an X-VIVO
medium, a STEM-LINE medium, and the like can be used. The
concentration of the treated serum in the medium is not
particularly limited, and is, for example, 0.01 to 20 v/v %,
preferably 0.1 to 10 v/v %, and more preferably 2.5 to 10 v/v %.
The number of hematopoietic stem cells per 1 ml of the medium is
1000 to 100000, for example. The medium further may contain
additives, and examples of the additives include cytokine,
antibiotics, salts, and vitamins.
[0041] The hematopoietic stem cells inoculated into the medium
containing the treated serum may be placed under ordinary culture
conditions for proliferating hematopoietic stem cells, for example.
According to the present invention, since the medium contains the
treated serum as described above, the proliferation and
differentiation of the hematopoietic stem cells can be inhibited by
culturing them in the medium. The temperature at the time of
culturing is 37.degree. C., for example. Furthermore, the
hematopoietic stem cells may be placed in the same medium for about
1 to 7 days, for example, and the medium preferably is replaced
with a new medium every 1 to 7 days. By such replacement of the
medium, it is possible to inhibit the proliferation and
differentiation of the hematopoietic stem cells for 1 to 7 days,
for example, and further, by accelerating the proliferation as will
be described below, it is also possible to start the proliferation.
A method for replacing the medium with a new medium is by no means
limited, and examples thereof include: replacing the medium at
predetermined times, as described above; and supplying a new medium
continuously or intermittently. When the latter is employed, it is
preferable to discard a part of the old medium continuously or
intermittently, for example.
[0042] In the present invention, the step (Y), i.e., the step of
accelerating the proliferation of hematopoietic stem cells, can be
carried out by inoculating the hematopoietic stem cells into a
medium containing non-sonicated serum, as described above. A
specific example thereof will be described below. It is to be
noted, however, the present invention is by no means limited to
this specific example.
[0043] As the medium, it is possible to use the same medium as
described above except that it contains the untreated serum instead
of the treated serum. The concentration of the untreated serum in
the medium is not particularly limited, and is, for example, 0.01
to 20 v/v %, preferably 0.1 to 10 v/v %, and more preferably 2.5 to
10 v/v %. The number of hematopoietic stem cells per 1 ml of the
medium is, for example, 1000 to 100000. The medium further may
contain the same additive as described above. The additives are not
particularly limited and may be the same as described above.
[0044] The hematopoietic stem cells inoculated into the medium
containing the untreated serum may be subjected to ordinary culture
conditions for proliferating hematopoietic stem cells, for example.
The temperature at the time of culturing is 37.degree. C., for
example. Furthermore, the hematopoietic stem cells may be placed in
the same medium for about 1 to 7 days, for example, and the medium
preferably is replaced with a new medium every 1 to 7 days. By
culturing the hematopoietic stem cells in the medium containing the
untreated serum, the number of the cells inoculated initially can
be increased about 2- to 10-fold through 7 days of culturing, for
example.
[0045] In the present invention, for example, when the step (Y) is
performed after the step (X) of inhibiting the proliferation and
differentiation, i.e., when the proliferation and differentiation
are inhibited until a desired time and the proliferation is started
to be accelerated from a desired time, the medium containing the
treated serum may be replaced with the medium containing the
untreated serum. On the other hand, for example, when the step (Y)
is performed prior to the step (X) of inhibiting the proliferation
and differentiation, i.e., when hematopoietic stem cells are
proliferated, and differentiation and further proliferation of the
proliferated hematopoietic stem cells are inhibited until a desired
time, the medium containing the untreated serum may be replaced
with the medium containing the treated serum. When the step (X) and
the step (Y) are repeated, the medium may be replaced depending on
the purpose, as described above.
[0046] <Production Method>
[0047] The production method according to the present invention is,
as described above, a method for producing cord blood hematopoietic
stem cells, including the step of controlling proliferation of
hematopoietic stem cells by the control method according to the
present invention.
[0048] The production method according to the present invention is
characterized in that the control method according to the present
invention is used in the inhibition of proliferation and
differentiation of hematopoietic stem cells, and other conditions
and configurations are by no means limited. Unless otherwise
stated, it can be carried out in the same manner as the control
method according to the present invention.
[0049] The production method according to the present invention
includes the step (X) as the step of inhibiting proliferation and
differentiation of hematopoietic stem cells, and preferably further
includes the step of accelerating proliferation of the
hematopoietic stem cells. The acceleration step is, for example,
the step of accelerating the proliferation of the hematopoietic
stem cells by inoculating cord blood hematopoietic stem cells into
a medium containing a liquid component of blood, and preferably is
the step (Y) in the control method according to the present
invention.
(X) inhibiting proliferation and differentiation of hematopoietic
stem cells by culturing the hematopoietic stem cells in a medium
containing a sonicated liquid component of cord blood (Y)
accelerating proliferation of the hematopoietic stem cells by
culturing the hematopoietic stem cells in a medium containing a
non-sonicated liquid component of cord blood
[0050] In the present invention, the medium used in the
acceleration step is not particularly limited, and preferably is a
medium containing a liquid component, for example. The liquid
component contained in the medium is not particularly limited, and
examples thereof include human cord blood serum, fetal bovine
serum, and human peripheral blood serum. The liquid component
preferably is, for example, a non-sonicated liquid component, more
preferably a non-sonicated liquid component of cord blood as used
in the above-described step (Y), and particularly preferably an
untreated liquid component derived from the same cord blood as the
hematopoietic stem cells.
[0051] In the present invention, either the inhibition step or the
proliferation acceleration step may be performed first, as in the
control method according to the present invention. The production
method according to a first embodiment is such that, for example,
first, the hematopoietic stem cells are inoculated into a medium
containing the treated liquid component and proliferation and
differentiation of the hematopoietic stem cells are inhibited until
a desired time at which the proliferation of the hematopoietic stem
cells is started, and proliferation of the hematopoietic stem cells
is started at the desired time. The production method according to
a second embodiment is such that, for example, first, the
hematopoietic stem cells are proliferated, and then the
proliferated hematopoietic stem cells are inoculated into a medium
containing the treated liquid component to inhibit the
proliferation and differentiation of the hematopoietic stem cells
until a desired time at which the hematopoietic stem cells are
used. It is possible to switch between the inhibition of the
proliferation and differentiation and the acceleration of the
proliferation by, for example, replacing the medium containing the
treated liquid component and the medium containing the untreated
liquid component depending on the purpose of the control, as in the
case of the above-described control method according to the present
invention, for example.
[0052] In the present invention, it is preferable to use
hematopoietic stem cells and a liquid component derived from cord
blood of the same individual, for example, as in the
above-described control method. Specifically, it is preferable to
inhibit the proliferation and differentiation using hematopoietic
stem cells and a treated liquid component derived from cord blood
of the same individual, and then accelerate the proliferation using
an untreated liquid component derived from cord blood of the same
individual. The individual is not particularly limited, and
examples thereof include humans and mammals excluding humans.
[0053] <Proliferation-Controlling Agent>
[0054] The proliferation-controlling agent according to the present
invention is for use in the control method according to the present
invention. The proliferation-controlling agent contains:
(a) a proliferation inhibitor for inhibiting proliferation and
differentiation of the hematopoietic stem cells, containing a
sonicated liquid component of cord blood.
[0055] It is only necessary that the proliferation-controlling
agent according to the present invention contains the sonicated
cord blood serum, and other configurations are by no means limited.
The proliferation-controlling agent according to the present
invention also can be used in the method for producing cord blood
hematopoietic stem cells according to the present invention, for
example.
[0056] The proliferation-controlling agent according to the present
invention further may contain a proliferation accelerator (b) shown
below. The proliferation inhibitor (a) and the proliferation
accelerator (b) exhibit opposite effects, i.e., inhibition and
acceleration. Thus, they are contained in a
proliferation-controlling agent separately as independent agents,
for example. (b) a proliferation accelerator for accelerating
proliferation of hematopoietic stem cells, containing a
non-sonicated liquid component of cord blood
[0057] In the proliferation-controlling agent according to the
present invention, it is preferable that the liquid component in
the proliferation inhibitor and the liquid component in the
proliferation accelerator are derived from cord blood of the same
individual. In the proliferation-controlling agent according to the
present invention, the treated liquid component in the
proliferation inhibitor and the untreated liquid component in the
proliferation accelerator are the same as described above, unless
otherwise stated.
[0058] <Proliferation-Controlling Kit>
[0059] The proliferation-controlling kit according to the present
invention is for use in the control method according to the present
invention. The proliferation-controlling kit includes:
(a) a proliferation inhibitor for inhibiting proliferation and
differentiation of the hematopoietic stem cells, containing a
sonicated liquid component of cord blood.
[0060] It is only necessary that the proliferation-controlling kit
according to the present invention includes the proliferation
inhibitor, and the proliferation inhibitor is the same as that used
in the proliferation-controlling agent according to the present
invention. The proliferation-controlling kit according to the
present invention also can be used in the method for producing cord
blood hematopoietic stem cells according to the present invention,
for example.
[0061] The proliferation-controlling kit according to the present
invention further may include a proliferation accelerator (b) shown
below, for example. By including the proliferation accelerator, the
proliferation-controlling kit easily can accelerate the
proliferation and inhibit the proliferation and differentiation of
the cord blood hematopoietic stem cells. The proliferation
accelerator is the same as that used in the
proliferation-controlling agent according to the present
invention.
(b) a proliferation accelerator for accelerating proliferation of
hematopoietic stem cells, containing a non-sonicated liquid
component of cord blood.
[0062] In the proliferation-controlling kit according to the
present invention, the proliferation inhibitor and the
proliferation accelerator preferably are contained in different
containers, because they are used for opposite purposes.
[0063] <Cord Blood Component Preparation Device>
[0064] The cord blood component preparation device according to the
present invention is a device for preparing cord blood
hematopoietic stem cells, a sonicated liquid component of cord
blood, and a non-sonicated liquid component of cord blood from the
same cord blood, for use in the control method or the production
method according to the present invention. According to the cord
blood component preparation device of the present invention, cord
blood hematopoietic stem cells to be controlled, a liquid component
for inhibiting the proliferation and differentiation, and a liquid
component for accelerating the proliferation can be prepared using
this device alone. First to fourth cord blood component preparation
devices according to the present invention will be described
below.
[0065] The first cord blood component preparation device of the
present invention includes:
[0066] a storage unit for storing cord blood;
[0067] an erythrocyte accommodation unit for accommodating
erythrocytes;
[0068] a hematopoietic stem cell accommodation unit for
accommodating hematopoietic stem cells;
[0069] first and second plasma accommodation units for
accommodating plasma; and
[0070] first and second serum accommodation units for accommodating
serum.
[0071] In the first cord blood component preparation device, the
storage unit is connected to each of the erythrocyte accommodation
unit, the hematopoietic stem cell accommodation unit, and the first
and second plasma accommodation units,
[0072] the first plasma accommodation unit is connected to the
first serum accommodation unit,
[0073] the second plasma accommodation unit is connected to the
second serum accommodation unit,
[0074] the storage unit is configured so that an erythrocyte
sedimenting agent can be introduced thereto,
[0075] the erythrocyte accommodation unit is configured so that
erythrocytes having sedimented from cord blood in the storage unit
can be introduced thereto,
[0076] the hematopoietic stem cell accommodation unit is configured
so that hematopoietic stem cells having sedimented from a
supernatant obtained after the sedimentation of the erythrocytes in
the storage unit can be introduced thereto,
[0077] the first plasma accommodation unit and the second plasma
accommodation unit are configured so that the supernatant obtained
after the sedimentation of the erythrocytes and the supernatant
obtained after the sedimentation of the hematopoietic stem cells in
the storage unit can be introduced thereto, respectively,
[0078] the first plasma accommodation unit is configured so that
sonication and a fibrin-forming treatment with respect to the
supernatant introduced thereto can be performed therein,
[0079] the first serum accommodation unit is configured so that,
among a sediment fraction and a supernatant fraction obtained from
the supernatant after being subjected to the sonication and the
fibrin-forming treatment in the first plasma accommodation unit,
the supernatant fraction can be introduced thereto as sonicated
serum,
[0080] the second plasma accommodation unit is configured so that a
fibrin-forming treatment with respect to the supernatant introduced
thereto can be performed therein, and
[0081] the second serum accommodation unit is configured so that,
among a sediment fraction and a supernatant fraction obtained from
the supernatant after being subjected to the fibrin-forming
treatment in the second plasma accommodation unit, the supernatant
fraction can be introduced thereto as non-sonicated serum.
[0082] The second cord blood component preparation device of the
present invention includes:
[0083] a storage unit for storing cord blood;
[0084] a cell-containing plasma accommodation unit for
accommodating plasma containing hematopoietic stem cells;
[0085] a hematopoietic stem cell accommodation unit for
accommodating hematopoietic stem cells;
[0086] first and second plasma accommodation units for
accommodating plasma; and
[0087] first and second serum accommodation units for accommodating
serum.
[0088] In the second cord blood component preparation device, the
storage unit is connected to the cell-containing plasma
accommodation unit,
[0089] the cell-containing plasma accommodation unit is connected
to each of the hematopoietic stem cell accommodation unit and the
first and second plasma accommodation units,
[0090] the first plasma accommodation unit is connected to the
first serum accommodation unit,
[0091] the second plasma accommodation unit is connected to the
second serum accommodation unit,
[0092] the storage unit is configured so that an erythrocyte
sedimenting agent can be introduced thereto,
[0093] the cell-containing plasma accommodation unit is configured
so that, among a sediment fraction containing erythrocytes having
sedimented from cord blood in the storage unit and a supernatant
fraction, the supernatant fraction can be introduced thereto as
plasma containing hematopoietic stem cells,
[0094] the hematopoietic stem cell accommodation unit is configured
so that hematopoietic stem cells having sedimented from the plasma
containing hematopoietic stem cells in the cell-containing plasma
accommodation unit can be introduced thereto,
[0095] the first plasma accommodation unit and the second plasma
accommodation unit are configured so that a supernatant obtained
after removing the hematopoietic stem cells in the cell-containing
plasma accommodation unit can be introduced thereto,
[0096] the first plasma accommodation unit is configured so that
sonication and a fibrin-forming treatment with respect to the
supernatant introduced thereto can be performed therein,
[0097] the first serum accommodation unit is configured so that,
among a sediment fraction and a supernatant fraction obtained from
the supernatant after being subjected to the sonication and the
fibrin-forming treatment in the first plasma accommodation unit,
the supernatant fraction can be introduced thereto as sonicated
serum,
[0098] the second plasma accommodation unit is configured so that a
fibrin-forming treatment with respect to the supernatant introduced
thereto can be performed therein, and
[0099] the second serum accommodation unit is configured so that,
among a sediment fraction and a supernatant fraction obtained from
the supernatant after being subjected to the fibrin-forming
treatment in the second plasma accommodation unit, the supernatant
fraction can be introduced thereto as non-sonicated serum.
[0100] The third cord blood component preparation device of the
present invention includes:
[0101] a storage unit for storing cord blood;
[0102] a cell-containing plasma accommodation unit for
accommodating plasma containing hematopoietic stem cells;
[0103] first and second plasma accommodation units for
accommodating plasma; and
[0104] first and second serum accommodation units for accommodating
serum.
[0105] In the third cord blood component preparation device, the
storage unit is connected to the cell-containing plasma
accommodation unit,
[0106] the cell-containing plasma accommodation unit is connected
to each of the first and second plasma accommodation units,
[0107] the first plasma accommodation unit is connected to the
first serum accommodation unit,
[0108] the second plasma accommodation unit is connected to the
second serum accommodation unit,
[0109] the storage unit is configured so that an erythrocyte
sedimenting agent can be introduced thereto,
[0110] the cell-containing plasma accommodation unit is configured
so that, among a sediment fraction containing erythrocytes having
sedimented from cord blood in the storage unit and a supernatant
fraction, the supernatant fraction can be introduced thereto as a
plasma fraction containing hematopoietic stem cells, and after the
plasma fraction introduced thereto is separated into a sediment
fraction containing the hematopoietic stem cells and a supernatant
fraction, the cell-containing plasma accommodation unit can be
divided into a lower region containing the sediment fraction and an
upper region containing the supernatant fraction,
[0111] the lower region of the cell-containing plasma accommodation
unit serves as a hematopoietic stem cell accommodation unit,
[0112] the first plasma accommodation unit and the second plasma
accommodation unit are configured so that the supernatant fraction
in the upper region of the cell-containing plasma accommodation
unit can be introduced thereto,
[0113] the first plasma accommodation unit is configured so that
sonication and a fibrin-forming treatment with respect to the
supernatant introduced thereto can be performed therein,
[0114] the first serum accommodation unit is configured so that,
among a sediment fraction and a supernatant fraction obtained from
the supernatant after being subjected to the sonication and the
fibrin-forming treatment in the first plasma accommodation unit,
the supernatant fraction can be introduced thereto as a sonicated
serum fraction,
[0115] the second plasma accommodation unit is configured so that a
fibrin-forming treatment with respect to the supernatant introduced
thereto can be performed therein, and
[0116] the second serum accommodation unit is configured so that,
among a sediment fraction and a supernatant fraction obtained from
the supernatant after being subjected to the fibrin-forming
treatment in the second plasma accommodation unit, the supernatant
fraction can be introduced thereto as non-sonicated serum.
[0117] The fourth cord blood component preparation device of the
present invention includes:
[0118] first and second storage units for storing cord blood;
[0119] an erythrocyte accommodation unit for accommodating
erythrocytes;
[0120] a hematopoietic stem cell accommodation unit for
accommodating hematopoietic stem cells;
[0121] first and second plasma accommodation units for
accommodating plasma; and
[0122] first, second, and third serum accommodation units for
accommodating serum.
[0123] In the fourth cord blood component preparation device, the
first storage unit is connected to each of the erythrocyte
accommodation unit, the hematopoietic stem cell accommodation unit,
and the first and second plasma accommodation units,
[0124] the first plasma accommodation unit is connected to the
first serum accommodation unit,
[0125] the second plasma accommodation unit is connected to the
second serum accommodation unit,
[0126] the first storage unit is configured so that an
anticoagulant and an erythrocyte sedimenting agent can be
introduced thereto,
[0127] the erythrocyte accommodation unit is configured so that
erythrocytes having sedimented from cord blood in the storage unit
can be introduced thereto,
[0128] the hematopoietic stem cell accommodation unit is configured
so that hematopoietic stem cells having sedimented from a
supernatant obtained after the sedimentation of the erythrocytes in
the storage unit can be introduced thereto,
[0129] the first plasma accommodation unit and the second plasma
accommodation unit are configured so that the supernatant obtained
after the sedimentation of the erythrocytes and the supernatant
obtained after the sedimentation of the hematopoietic stem cells in
the storage unit can be introduced thereto, respectively,
[0130] the first plasma accommodation unit is configured so that
sonication and a fibrin-forming treatment with respect to the
supernatant introduced thereto can be performed therein,
[0131] the first serum accommodation unit is configured so that,
among a sediment fraction and a supernatant fraction obtained from
the supernatant after being subjected to the sonication and the
fibrin-forming treatment in the first plasma accommodation unit,
the supernatant fraction can be introduced thereto as sonicated
serum,
[0132] the second plasma accommodation unit is configured so that a
fibrin-forming treatment with respect to the supernatant introduced
thereto can be performed therein,
[0133] the second serum accommodation unit is configured so that,
among a sediment fraction and a supernatant fraction obtained from
the supernatant after being subjected to the fibrin-forming
treatment in the second plasma accommodation unit, the supernatant
fraction can be introduced thereto as non-sonicated serum,
[0134] the second storage unit is connected to the third serum
accommodation unit;
[0135] the second storage unit is configured so that a blood
coagulation-accelerating substance for accelerating blood
coagulation can be introduced thereto, and
[0136] the third serum accommodation unit is configured so that a
supernatant fraction obtained after blood coagulation in the second
storage unit can be introduced thereto.
[0137] In the present invention, it is preferable that the storage
unit and the respective accommodation units are connected to each
other in an airtight manner. This allows the inside of the device
of the present invention to be kept sterile, for example, and a
series of operations subsequent to the introduction of cord blood
can be carried out without exposing the cord blood to the outside
air, for example. Furthermore, by connecting the storage unit and
the respective accommodation units in an airtight manner, they also
can be kept in a liquid-tight state, for example.
[0138] It is preferable that the storage unit and the respective
accommodation units are formed of a flexible material, for example.
Examples of the flexible material include: polyolefins such as
polyethylene, polypropylene, polytetrafluoroethylene, and
polybutylene terephthalate; polyvinyl chlorides such as soft
polyvinyl chloride; polyvinyl alcohols; ethylene-vinyl acetate
copolymer resin; silicone; polyurethane; and resins such as nylon.
Furthermore, it is preferable that the cord blood component
preparation device according to the present invention can be
sterilized, for example, from a hygiene viewpoint, and, as will be
described below, the respective plasma accommodation units may be
subjected to a heat treatment as the fibrin-forming treatment, for
example. Thus, the flexible material preferably has heat
resistance.
[0139] The fibrin-forming treatment in the first plasma
accommodation unit and the second plasma accommodation unit is not
particularly limited, and examples thereof include: a heat
treatment to the plasma accommodation units; and introduction of a
blood coagulation reagent to the plasma accommodation units. In the
former case, the respective plasma accommodation units may be
configured so that a heat treatment to the supernatant introduced
thereto can be performed, for example. In the latter case, the
respective plasma accommodation units may be configured so that the
blood coagulation reagent can be introduced thereto, for example.
The blood coagulation reagent may be accommodated in the respective
plasma accommodation units in advance, or may be introduced to the
respective plasma accommodation units when used, for example. The
fibrin-forming treatment in the first and second plasma
accommodation units of the present invention preferably is a heat
treatment, for example.
[0140] The form of the storage unit and the respective
accommodation units is not particularly limited. Preferably, they
are in the form of bag from the aspect of excellent handleability,
for example. The bag can be formed by, for example, laminating two
flexible resin sheets and then bonding an edge portion (seal
portion) as the rim of the laminate. The type of the bonding is not
particularly limited, and examples thereof include: welding such as
heat-sealing and ultrasonic welding; and adhesion using an adhesive
or the like.
[0141] The size of the storage unit and the respective
accommodation units is not particularly limited. The capacity of
the storage unit preferably is 50 to 400 ml, for example, and the
amount of cord blood to be introduced to the storage unit
preferably is 40 to 80 vol % of the capacity of the storage unit,
for example. The capacities of the respective accommodation units
can be determined as appropriate depending on, for example, the
amount of cord blood that can be introduced to the storage unit. It
preferably is 25 to 200 ml, and preferably is 30 to 70 vol % of the
capacity of the storage unit. The storage unit and the respective
accommodation units may contain air inside, for example. However,
it is preferable that the content of the air is as small as
possible. In the fourth cord blood component preparation device,
the capacity of the first storage unit is the same as described
above, for example, and the capacity of the second storage unit
preferably is 10 to 80 vol %, for example, and the amount of cord
blood to be introduced to the second storage unit preferably is 40
to 80 vol % of the capacity of the second storage unit, for
example.
[0142] It is preferable that the storage unit and the respective
accommodation units are connected to each other through tubes, for
example. The tubes preferably are formed of a flexible material,
for example. Furthermore, as will be described below, it is
preferable that the hematopoietic stem cell accommodation unit 12,
the first serum accommodation unit 13b, and the second serum
accommodation unit 14b are detached from the cord blood component
preparation device in the state where the flow paths of the tubes
are blocked. Thus, the tubes preferably are formed of a material
that can be cut and fused by heating and melting, for example.
Examples of such a material include: polyolefins such as
polyethylene, polypropylene, polytetrafluoroethylene, and
polybutylene terephthalate; polyvinyl chloride such as soft
polyvinyl chloride; polyvinyl alcohols; ethylene-vinyl acetate
copolymer resins; silicone; polyurethane; and nylon. Preferably,
the cord blood component preparation device of the present
invention further includes a member that can block and unblock the
flow path of a tube as appropriate, for example. Examples of the
member include clamping members such as a clamp and forceps. By
pinching each tube with the clamping member as appropriate, it is
possible to switch the flow path between the storage unit and the
respective accommodation units and between the respective
accommodation units. Furthermore, as the member that opens/closes
the flow path, it is also possible to use a valve or the like, for
example. The valve may be disposed at a certain point on the tube,
for example, or may be provided at a branched portion of the tubes,
which will be described below. Furthermore, a branch connector to
be described below also may serve as a valve.
[0143] In the first and third cord blood component preparation
devices, for example, when cord blood is introduced to the storage
unit directly from a patient, the storage unit further may contain
an anticoagulant or may be configured so that the anticoagulant can
be introduced thereto. In the latter case, for example, it is
preferable to introduce the anticoagulant to the storage unit
before introducing cord blood to the storage unit. The
anticoagulant is not particularly limited, and examples thereof
include a CPD solution (Citrate-Phosphate-Dextrose solution) and an
ACD-A solution (Acid-Citrate-Dextrose-A solution). The amount of
the anticoagulant is not particularly limited, and can be
determined depending on the volume of the storage unit, the volume
of the cord blood to be introduced to the storage unit, and the
like, for example. The amount of the anticoagulant to be added
preferably is 30 to 100 vol % of cord blood to be introduced, for
example. In the fourth cord blood component preparation device, an
anticoagulant can be introduced to the first storage unit as
described above, and it is not necessary to introduce an
anticoagulant to the second storage unit.
[0144] In the first to third cord blood component preparation
devices, an erythrocyte sedimenting agent may be accommodated in
the storage unit in advance, or an erythrocyte sedimenting agent
may be introduced to the storage unit from the outside, for
example. For example, in the case where cord blood containing an
erythrocyte sedimenting agent is introduced to the storage unit, it
is not necessary that the erythrocyte sedimenting agent is
accommodated in or introduced to the storage unit.
[0145] The cord blood component preparation device according to the
present invention can be used for preparing components not only
from human cord blood but also from cord blood of mammals such as
rodents, domestic animals, and primates, for example.
First Embodiment
[0146] Next, an example of the first cord blood component
preparation device according to the present invention will be
described with reference to FIG. 1. It is to be noted, however, the
present invention is not limited thereto.
[0147] FIG. 1 is a plan view schematically showing the cord blood
component preparation device of the present embodiment. The cord
blood component preparation device 1 includes: a storage unit 10
for storing cord blood; an erythrocyte accommodation unit 11 for
accommodating erythrocytes; a hematopoietic stem cell accommodation
unit 12 for accommodating hematopoietic stem cells; a first plasma
accommodation unit 13a and a second plasma accommodation unit 14a
for accommodating plasma; and a first serum accommodation unit 13b
and a second serum accommodation unit 14b for accommodating serum.
In the storage unit 10 and the respective accommodation units 11 to
14b shown in FIG. 1, the inside of the dotted line is a space
capable of accommodating the respective components, and the portion
between the dotted line and the solid line indicating the outer
frame is a seal portion. The storage unit 10 has two inlets 15a and
15b and two outlets 16c and 16f. The left inlet 15a of the storage
unit 10 is an inlet for cord blood, and one end of a tube 18a is
connected thereto. The other end of the tube 18a is a connection
part 9 to a cord blood bag. For example, a puncture needle or the
like can be connected thereto, and the connection part 9 is covered
with a cap or the like until it is used. The right inlet 15b of the
storage unit 10 is an inlet for an erythrocyte sedimenting agent,
and one end of a tube 18b is connected thereto. The other end of
the tube 18b is a connection part 9 to a container of an
erythrocyte sedimenting agent, and the connection part 9 is covered
with a cap or the like until it is used. The lower outlet 16c of
the storage unit 10 is an outlet for erythrocytes and hematopoietic
stem cells having sedimented from the cord blood, and one end of a
tube 18c is connected thereto. The upper outlet 16f is an outlet
for plasma obtained after removing the erythrocytes and the
hematopoietic stem cells, and one end of a tube 18f is connected
thereto. The erythrocyte accommodation unit 11 has an inlet 15d for
the erythrocytes having sedimented from the cord blood, and one end
of a tube 18d is connected to the inlet 15d. The hematopoietic stem
cell accommodation unit 12 has an inlet 15e for the hematopoietic
stem cells having sedimented from the cord blood, and one end of a
tube 18e is connected to the inlet 15e. The tube 18c connected to
the storage unit 10, the tube 18d connected to the erythrocyte
accommodation unit 11, and the tube 18e connected to the
hematopoietic stem cell accommodation unit 12 communicate with each
other with the other ends thereof being connected to each other via
a branch connector 19. The first plasma accommodation unit 13a has
an inlet 15g for the plasma obtained after removing the
erythrocytes and the hematopoietic stem cells and an outlet 16i for
serum. One end of a tube 18g is connected to the inlet 15g, and one
end of a tube 18i is connected to the outlet 16i. On the other
hand, the second plasma accommodation unit 14a also has an inlet
15h for the plasma obtained after removing the erythrocytes and the
hematopoietic stem cells and an outlet 16k for serum. One end of a
tube 18h is connected to the inlet 15h, and one end of a tube 18k
is connected to the outlet 16k. The tube 18f connected to the
storage unit 10, the tube 18g connected to the first plasma
accommodation unit 13a, and the tube 18h connected to the second
plasma accommodation unit 14a communicate with each other with the
other ends thereof being connected to each other via another branch
connector 19. The first serum accommodation unit 13b has an inlet
15i for the serum, and the other end of the tube 18i connected to
the first plasma accommodation unit 13a is connected to the inlet
15i. The second serum accommodation unit 14b has an inlet 15k for
the serum, and the other end of the tube 18k connected to the
second plasma accommodation unit 14a is connected to the inlet
15k.
[0148] The cord blood component preparation device 1 preferably has
clamping members (not shown), such as clamps, for clamping the
tubes, for example. Preferably, the tubes connecting the storage
unit 10 to the respective accommodation units 11, 12, 13a, and 14a,
and the tubes connecting the plasma accommodation units 13a and 14a
to the serum accommodation units 13b and 14b are provided with the
clamping members, for example. With the use of the clamping
members, switching to a desired flow path becomes possible.
[0149] Between the first plasma accommodation unit 13a and the
first serum accommodation unit 13b (e.g., the tube 18i) and between
the second plasma accommodation unit 14a and the second serum
accommodation unit 14b (e.g., the tube 18k), filters for removing
remaining erythrocytes and the like may be present, for example.
Such a filter may be present also in the tube 18h connected to the
second plasma accommodation unit 14a.
[0150] Each of the accommodation units may have an openable outlet
17, for example. For example, when separating various components of
the cord blood, the opening of the outlet 17 preferably is closed
as shown in FIG. 1. Then, when discharging the various components
accommodated in the respective accommodation units to the outside,
the components inside can be discharged through the outlet 17
opened by removing the closed portion. Also, by inserting a
collection needle into the closed outlet 17, the components inside
can be discharged through the collection needle, for example.
[0151] A method for preparing hematopoietic stem cells, sonicated
serum (treated serum), and non-sonicated serum (untreated serum)
from cord blood using the cord blood component preparation device 1
of the present embodiment will be described with reference to an
illustrative example. Specific treatment conditions can be set to
be the same as those described above, unless otherwise stated.
[0152] First, cord blood is provided. In the case of human cord
blood, it is available in the state of being accommodated in a
blood bag, for example. Human cord blood accommodated in a blood
bag generally contains an anticoagulant. To this blood bag, one end
of the tube 18a connected to the storage unit 10 is connected, and
the cord blood is introduced to the storage unit 10. A blood
collection needle may be attached to the connection part 9 provided
at the tip of the tube 18a, and the cord blood may be introduced to
the storage unit 10 by inserting the blood collection needle into
the blood bag, for example. At this time, in order to prevent the
cord blood in the storage unit 10 from flowing into the tubes 18c,
18d, and 18e, the erythrocyte accommodation unit 11, and the
hematopoietic stem cell accommodation unit 12 via the lower outlet
16c, it is preferable to block a flow path of the tube 18c with a
clamp in the vicinity of the storage unit 10. After the cord blood
is introduced to the storage unit 10, the tip of the tube 18a for
cord blood introduction may be covered with a cap, or a desired
portion of the tube 18a may be cut and fused using a sealer or the
like, for example. Also, cord blood may be collected directly from
a patient through the blood collection needle attached to the tip
of the tube 18a and stored in the storage unit 10. When cord blood
is collected directly from a patient and stored in the storage unit
10 as described above, an anticoagulant may be accommodated in the
storage unit 10 in advance, for example. Alternatively, prior to
the introduction of cord blood, an anticoagulant may be introduced
to the storage unit 10 through the tube 18b connected to the
storage unit 10, for example.
[0153] Subsequently, an erythrocyte sedimenting agent is introduced
to the storage unit 10 through the tube 18b connected to the
storage unit 10, and the cord blood is mixed with the erythrocyte
sedimenting agent. Thereafter, the storage unit 10 was allowed to
stand still. Then, after the cord blood has separated into an
erythrocyte fraction and a supernatant fraction in the storage unit
10, the erythrocyte fraction having sedimented is introduced to the
erythrocyte accommodation unit 11 through the tubes 18c and 18d
under the storage unit 10. At this time, in order to prevent the
erythrocyte fraction from being introduced to the hematopoietic
stem cell accommodation unit 12, it is preferable to block the flow
path of the tube 18e connected to the hematopoietic stem cell
accommodation unit 12 with a clamp in the vicinity of the branch
connector 19 and then release the blocking of the flow path of the
tube 18c with the clamp. This opens the flow path extending from
the storage unit 10 to the erythrocyte accommodation unit 11,
thereby allowing the erythrocyte fraction to be introduced to the
erythrocyte accommodation unit 11. After the erythrocyte fraction
is introduced to the erythrocyte accommodation unit 11, it is
preferable to block the flow path of the tube 18c again with a
clamp in the vicinity of the storage unit 10.
[0154] After the erythrocyte fraction is discharged in the above
described manner, the storage unit 10 is centrifuged, thus
achieving the separation into a sediment fraction containing
hematopoietic stem cells and a supernatant fraction containing
plasma. At this time, conditions for the centrifugation are as
described above.
[0155] Then, through the tubes 18c and 18e under the storage unit
10, the hematopoietic stem cell fraction having sedimented is
introduced to the hematopoietic stem cell accommodation unit 12. At
this time, in order to prevent the hematopoietic stem cell fraction
from being introduced to the erythrocyte accommodation unit 11, it
is preferable to block the flow path of the tube 18d connected to
the erythrocyte accommodation unit 11 with a clamp in the vicinity
of the branch connector 19 and then release the blocking of the
flow path of the tube 18c with the clamp. After the hematopoietic
stem cell fraction is introduced to the hematopoietic stem cell
accommodation unit 12, it is preferable to block the flow path of
the tube 18c again with a clamp in the vicinity of the storage unit
10. Alternatively, a desired portion of the tube 18c or the inlet
15e may be cut and fused using a sealer or the like. By this
operation, a sealed hematopoietic stem cell-accommodating bag
accommodating the hematopoietic stem cell fraction is obtained from
the cord blood component preparation device 1. The hematopoietic
stem cells in the bag can be taken out from the bag by cutting off
the blocked portion of the outlet 17 or piercing the blocked outlet
17 with a collection needle, for example.
[0156] On the other hand, the plasma fraction remaining in the
storage unit 10 is dispensed to the first plasma accommodation unit
13a and the second plasma accommodation unit 14a through the tubes
18f, 18g, and 18h. When introducing the plasma fraction to the
first plasma accommodation unit 13a and then to the second plasma
accommodation unit 14a, it is preferable to introduce the plasma
fraction to the first plasma accommodation unit 13a with the flow
path of the tube 18h connected to the second plasma accommodation
unit 14a being blocked in the vicinity of the branch connector 19,
then block the flow path of the tube 18g connected to the first
plasma accommodation unit 13a in the vicinity of the branch
connector 19, and thereafter, to release the blocking of the flow
path of the tube 18h with the clamp, thereby allowing the plasma
fraction to be introduced to the second plasma accommodation unit
14a, for example.
[0157] Next, the first plasma accommodation unit 13a is subjected
to sonication. The sonication can be carried out by immersing the
plasma accommodation unit and an oscillator in a liquid in a bath
and causing the oscillator to output ultrasonic waves, for example.
Subsequently, the first plasma accommodation unit 13a is subjected
to a heat treatment. By this heat treatment, fibrin is formed from
fibrinogen in the plasma fraction. The heat treatment can be
carried out by immersing the first plasma accommodation unit 13a in
a heated liquid in a bath, for example. The first plasma
accommodation unit 13a after being subjected to the heat treatment
is centrifuged, thus achieving the separation into a sediment
fraction containing the formed fibrin, platelets, and the like and
a supernatant fraction containing serum. This supernatant fraction
is introduced to the first serum accommodation unit 13b through the
tube 18i connecting the first plasma accommodation unit 13a to the
first serum accommodation unit 13b. In this manner, the sonicated
serum can be collected. When introducing the supernatant fraction
in the first plasma accommodation unit 13a to the first serum
accommodation unit 13b, it is preferable to prevent the sediment
fraction from entering the first serum accommodation unit 13b. In
this case, examples of the method of preventing this include
pinching the boundary between the supernatant fraction and the
sediment fraction with a clamp or the like from the outside of the
first plasma accommodation unit 13a so as to separate the
supernatant fraction and the sediment fraction.
[0158] On the other hand, the second plasma accommodation unit 14a
is subjected to a heat treatment in the same manner as in the
above, except that the second plasma accommodation unit 14a is not
subjected to sonication and that platelets are removed. The heat
treatment of the second plasma accommodation unit 14a may be
performed at the same time as the heat treatment of the first
plasma accommodation unit 13a, for example. Subsequently, the
second plasma accommodation unit 14a after being subjected to the
heat treatment is centrifuged, thus achieving the separation into a
sediment fraction containing the formed fibrin, platelets, and the
like and a supernatant fraction containing serum. This supernatant
fraction is introduced to the second serum accommodation unit 14b
through the tube 18k connecting the second plasma accommodation
unit 14a to the second serum accommodation unit 14b. In this
manner, non-sonicated serum can be collected.
[0159] With regard to the first serum accommodation unit 13b and
the second serum accommodation unit 14b, a desired portion of the
tube 18i or the inlet 15i and a desired portion of the tube 18k or
the inlet 15k may be cut and fused using a sealer or the like, for
example. By this operation, a sealed serum-accommodating bag in
which the treated serum is accommodated and a sealed
serum-accommodating bag in which the untreated serum is
accommodated can be obtained from the cord blood component
preparation device 1. The serum in each of the bags can be taken
out from the bag by cutting off the blocked portion of the outlet
17 or piercing the blocked outlet 17 with a collection needle, for
example.
[0160] The hematopoietic stem cell bag accommodating the
hematopoietic stem cells, serum bag accommodating the treated
serum, and serum bag accommodating the untreated serum obtained in
the above-described manner can be preserved by cryopreservation or
the like before use, for example. When preserving them by
cryopreservation, after the hematopoietic stem cell accommodation
unit 12, the first serum accommodation unit 13b, and the second
serum accommodation unit 14b have accommodated the components to be
accommodated therein, respectively, a cryoprotectant may be
introduced to these units through any of the tubes connected
thereto and then the tubes may be cut off and fused, for
example.
[0161] Furthermore, in the embodiment of the present invention,
when removing fibrinogen from plasma and collecting serum as
described above, it is possible to utilize the activation mechanism
of a blood coagulation system instead of the heat treatment. In
this case, it is preferable that, after the first plasma
accommodation unit 13a is subjected to sonication in a manner
described above, a blood coagulation reagent further is added to
the first plasma accommodation unit 13a. Examples of the blood
coagulation reagent include the above-described coagulation factor
such as thrombin. When the cord blood contains an anticoagulant, it
is preferable that the blood coagulation reagent further contains a
neutralizer such as calcium chloride, for example. By adding the
blood coagulation reagent as described above, fibrin is formed from
fibrinogen in plasma, so that the fibrinogen can be removed by
carrying out centrifugation out thereafter in the same manner as in
the above. The blood coagulation reagent may be added by adding a
coagulation factor such as thrombin and a neutralizer to the plasma
at the same time, or by adding the coagulation factor after adding
the neutralizer, for example. In such an aspect of the invention,
it is preferable that the first plasma accommodation unit 13a has
an inlet for the blood coagulation reagent. The inlet preferably is
configured so that, for example, one end of a tube is connected
thereto and the other end of the tube serves as a connection part
to an accommodation unit for the blood coagulation reagent, which
is covered with a cap or the like before use. Furthermore, it is
also preferable to add the blood coagulation reagent to the second
plasma accommodation unit 14a, instead of subjecting the second
plasma accommodation unit 14a to a heat treatment. It is also
preferable that the second plasma accommodation unit 14a has an
inlet for the blood coagulation reagent. The inlet preferably is
configured so that, for example, one end of a tube is connected
thereto and the other end of the tube serves as a connection part
to an accommodation unit for the blood coagulation reagent, which
is covered with a cap or the like before use.
Second Embodiment
[0162] Next, an example of the second cord blood component
preparation device according to the present invention will be
described with reference to FIG. 2. In FIG. 2, elements the same as
those in FIG. 1 are marked with the same reference numerals. The
second cord blood component preparation device is the same as the
first cord blood component preparation device, unless otherwise
stated. Unless otherwise stated, the present invention is not
limited thereto.
[0163] FIG. 2 is a plan view schematically showing the cord blood
component preparation device of the present embodiment. The cord
blood component preparation device 2 includes: a storage unit 10
for storing cord blood; a cell-containing plasma accommodation unit
20 for accommodating hematopoietic stem cell-containing plasma
obtained after removing erythrocytes; a hematopoietic stem cell
accommodation unit 12 for accommodating hematopoietic stem cells; a
first plasma accommodation unit 13a and a second plasma
accommodation unit 14a for accommodating plasma; and a first serum
accommodation unit 13b and a second serum accommodation unit 14b
for accommodating serum. The storage unit 10 has two inlets 15a and
15b and an outlet 16f. The left inlet 15a of the storage unit 10 is
an inlet for cord blood, and one end of a tube 18a is connected
thereto. The other end of the tube 18a is a connection part 9 to a
cord blood bag. The right inlet 15b of the storage unit 10 is an
inlet for an erythrocyte sedimenting agent, and one end of a tube
18b is connected thereto. The other end of the tube 18b is a
connection part 9 to a container of an erythrocyte sedimenting
agent. The upper outlet 16f of the storage unit 10 is an outlet for
hematopoietic stem cell-containing plasma obtained after removing
erythrocytes, and one end of a tube 21a is connected thereto. The
cell-containing plasma accommodation unit 20 has an inlet 25b and
an outlet 26c. The inlet 25b provided in the upper part is an inlet
for hematopoietic stem cell-containing plasma, and one end of a
tube 21b is connected thereto. The other end of the tube 21b
connected to the cell-containing plasma accommodation unit 20 is
connected to the other end of the tube 21a connected to the storage
unit 10 and one end of a tube 21d via a branch connector 19. The
other end of the tube 21d is connected to first ends of tubes 18g
and 18h connected to the first plasma accommodation unit 13a and
the second plasma accommodation units 14a via another branch
connector 19.
[0164] The cord blood component preparation device 2 preferably has
clamping members (not shown), such as clamps, for clamping the
tubes, for example. Preferably, the clamping member such as
described above is provided in: the tube 21a connecting the storage
unit 10 and the cell-containing plasma accommodation unit 20; the
tube 21d; the tube 21c connecting the cell-containing plasma
accommodation unit 20 and the hematopoietic stem cell accommodation
unit 12; the tubes 18g and 18h extending from the branch connector
19 and connected to the plasma accommodation units 13a and 14a,
respectively; and the tubes 18i and 18k connecting the plasma
accommodation units 13a and 14a to the serum accommodation units
13b and 14b, respectively, for example.
[0165] A method for preparing hematopoietic stem cells, treated
serum, and untreated serum from cord blood using the cord blood
component preparation device 2 of the present embodiment will be
described with reference to an illustrative example. Specific
treatment conditions can be set to be the same as those described
above, unless otherwise stated.
[0166] The cord blood in the storage unit 10 is caused to separate
into an erythrocyte fraction and a supernatant fraction in the same
manner as described above, and thereafter, the supernatant fraction
is introduced to the cell-containing plasma accommodation unit 20
through the tubes 21a and 21b extending from the upper part of the
storage unit 10. The supernatant fraction is a plasma fraction
containing hematopoietic stem cells. At this time, in order to
prevent the supernatant fraction from being introduced to the
plasma accommodation units 13a and 14a, it is preferable to block
the flow path of the tube 21d with a clamp in the vicinity of the
branch connector 19 connected to the tube 21b. Furthermore, in
order to prevent the supernatant fraction introduced to the
cell-containing plasma accommodation unit 20 from being discharged
to the hematopoietic stem cell accommodation unit 12, it is
preferable to block the flow path of the tube 21c with a clamp in
the vicinity of the outlet 26c.
[0167] Next, the cell-containing plasma accommodation unit 20 is
centrifuged, thus achieving the separation into a sediment fraction
containing hematopoietic stem cells and a supernatant fraction
containing plasma. At this time, conditions for the centrifugation
preferably are set so that, as described above, platelets are
contained in the supernatant fraction.
[0168] Then, through the tube 21c under the cell-containing plasma
accommodation unit 20, the sediment fraction containing
hematopoietic stem cells is introduced to the hematopoietic stem
cell accommodation unit 12. It is preferable that, after
introducing the hematopoietic stem cells, the flow path of the tube
21c connecting the cell-containing plasma accommodation unit 20 and
the hematopoietic stem cell accommodation unit 12 is blocked with a
clamp.
[0169] On the other hand, the plasma fraction remaining in the
cell-containing plasma accommodation unit 20 is dispensed to the
first plasma accommodation unit 13a and the second plasma
accommodation unit 14a through the tubes 21b, 21d, 18g, and 18h. At
this time, in order to prevent the plasma fraction from being
discharged to the storage unit 10, it is preferable to block the
flow path of the tube 21a connected to the storage unit 10 with a
clamp in the vicinity of the branch connector 19. Then, the plasma
dispensed to each unit is treated in the same manner as above so as
to prepare serum.
Third Embodiment
[0170] Next, an example of the third cord blood component
preparation device according to the present invention will be
described with reference to FIG. 3. In FIG. 3, elements the same as
those in FIGS. 1 and 2 are marked with the same reference numerals.
The third cord blood component preparation device is the same as
the first and second cord blood component preparation devices,
unless otherwise stated. Unless otherwise stated, the present
invention is not limited thereto.
[0171] FIG. 3 is a plan view schematically showing the cord blood
component preparation device of the present embodiment. The cord
blood component preparation device 3 is the same as the second cord
blood component preparation device described above, except that it
includes, instead of the cell-containing plasma accommodation unit
20 and the hematopoietic stem cell accommodation unit 12 shown in
FIG. 2, a dividable cell-containing plasma accommodation unit 30.
The cell-containing plasma accommodation unit 30 is configured so
that, among a sediment fraction containing erythrocytes having
sedimented from cord blood and a supernatant fraction in the
storage unit 10, the supernatant fraction can be introduced thereto
as a plasma fraction containing hematopoietic stem cells.
Furthermore, the cell-containing plasma accommodation unit 30 is
configured so that, after the plasma fraction introduced thereto is
caused to separate into a sediment fraction containing
hematopoietic stem cells and a supernatant fraction, the space
inside can be divided into a lower region 30b having the sediment
fraction and an upper region 30a having the supernatant
fraction.
[0172] After the hematopoietic stem cell-containing plasma obtained
after removing the erythrocytes is introduced to the
cell-containing plasma accommodation unit 30, the cell-containing
plasma accommodation unit 30 is centrifuged, thus achieving the
separation into a sediment fraction containing hematopoietic stem
cells and a supernatant fraction containing plasma. After the
separation, the boundary between the lower region 30b having the
sediment fraction and the upper region 30a having the supernatant
fraction is pinched with a gripping member from the outside of the
cell-containing plasma accommodation unit 30 so as to divide these
regions. Specifically, as shown in FIG. 4, for example, the
boundary X between the upper region 30a and the lower region 30b of
the cell-containing plasma accommodation unit 30 is pinched with a
clamp 40.
[0173] Then, the supernatant fraction accommodated in the upper
region 30a is discharged to plasma accommodation units 13a and 14a,
and serums are prepared in the same manner as above. On the other
hand, the cell-containing plasma accommodation unit 30 after the
supernatant fraction has been discharged therefrom (the lower
region 30b) serves as an accommodation unit for hematopoietic stem
cells.
[0174] In the third cord blood component preparation device 3, the
cell-containing plasma accommodation unit may be a cell-containing
plasma accommodation unit 50 shown in FIG. 5, for example. As shown
in FIG. 5, the cell-containing plasma accommodation unit 50 is
configured so that a lower part of an upper region 50a is tapered
so as to allow erythrocytes having sedimented therein to move to a
lower region 50b easily.
Fourth Embodiment
[0175] Next, the fourth cord blood component preparation device of
the present invention will be described. The fourth cord blood
component preparation device of the present invention is a device
to be used when preparing the respective components by introducing
uncoagulated cord blood that does not contain a blood coagulant,
for example. The uncoagulated cord blood that does not contain a
blood coagulant generally is cord blood introduced directly from a
patient. According to the cord blood component preparation device
of the present embodiment, it is possible to obtain serum in which
platelets are activated with a blood coagulation-accelerating
substance that accelerates blood coagulation, in addition to the
above-described cord blood stem cells, treated serum, and untreated
serum.
[0176] An example of the fourth cord blood component preparation
device according to the present invention will be described with
reference to FIG. 6. In FIG. 6, elements the same as those in FIGS.
1 to 5 are marked with the same reference numerals. The fourth cord
blood component preparation device is the same as the first to
third cord blood component preparation devices, unless otherwise
stated. Unless otherwise stated, the present invention is not
limited thereto.
[0177] FIG. 6 is a plan view schematically showing the cord blood
component preparation device of the present embodiment. The cord
blood component preparation device 6 is the cord blood component
preparation device of FIG. 1 illustrating the first embodiment,
further including a second storage unit 60 for storing cord blood
and a third serum accommodation unit 61b. The storage unit 10 in
FIG. 1 corresponds to a first storage unit in the present
embodiment. The first storage unit 10 is the same as the storage
unit in the first embodiment unless otherwise stated. Also, the
respective accommodation units connected to the first storage unit
10 through tubes and the like are the same as those in the first
embodiment illustrated in FIG. 1 unless otherwise stated. To the
first storage unit 10, a blood coagulation reagent and an
erythrocyte sedimenting agent can be introduced. The first storage
unit 10 has an inlet 15a for cord blood, and one end of a tube 68a
is connected to the inlet 15a. The second storage unit 60 can
accommodate a blood coagulation-accelerating substance (blood
coagulation accelerator) that accelerates blood coagulation. The
second storage unit 60 has an inlet 65a and an outlet 66a. The left
inlet 65a of the second storage unit 60 is an inlet for cord blood,
and one end of a tube 68b is connected thereto. The tube 68a
connected to the first storage unit 10 and the tube 68b connected
to the second storage unit 60 are connected to a tube 68c via a
branch connector 19. The other end of the tube 68c is a connection
part 9 to which a puncture needle or the like for collecting cord
blood can be connected. The right outlet 66a of the second storage
unit 60 is an outlet for discharging serum after blood coagulation
and a tube 68d is connected thereto. Furthermore, the third serum
accommodation unit 61b has an inlet 65b, and the other end of the
tube 68d connected to the second storage unit 60 is connected to
the inlet 65b.
[0178] In the cord blood component preparation device of the
present embodiment, as will be described below, a blood coagulation
cascade is caused to act by the contact of the blood
coagulation-accelerating substance with cord blood in the second
storage unit, thereby causing the coagulation of blood as well as
the release of growth factors by the activation of platelets. Thus,
in the second storage unit 60, a supernatant obtained after the
blood coagulation contains more growth factors as compared with the
case where platelets are not activated.
[0179] The blood coagulation-accelerating substance is not
particularly limited, and those described in Japanese Patent No.
3788479 and the like can be used, for example. The shape of the
blood coagulation-accelerating substance preferably is granular,
fine-granular, or massive, for example, and a substantially
spherical shape also is preferable. The size of the blood
coagulation-accelerating substance is not particularly limited, and
it is preferable that the diameter thereof is 1 to 10 mm, more
preferably 3 to 5 mm, for example. The blood
coagulation-accelerating substance may have a porous structure, for
example. When the blood coagulation-accelerating substance has a
porous structure, the area per unit volume thereof can be made
large, thus allowing the activation of platelets and the like to be
carried out efficiently.
[0180] The material of the blood coagulation-accelerating substance
is not particularly limited. Preferably, the surface of the blood
coagulation-accelerating substance is formed of a layer containing
a silicon dioxide, for example. Examples of the silicon dioxide
include glass, silica, diatomaceous earth, and kaoline.
Furthermore, the blood coagulation-accelerating substance may be
such that, for example, the core thereof contains a magnetic
substance, for example. When the blood coagulation-accelerating
substance contains a magnetic substance as described above, it is
possible to stir cord blood by, for example, applying a magnetic
field to the second storage unit in which the blood
coagulation-accelerating substance is accommodated, whereby rapid
activation of platelets and the like becomes possible.
[0181] The amount of the blood coagulation-accelerating substance
in the second storage unit is not particularly limited, and can be
determined as appropriate depending on the amount of cord blood
that the storage unit can accommodate, for example. As a specific
example, it is preferable to set the total surface area of the
blood coagulation-accelerating substance to 0.1 to 25 mm.sup.2/ml
with respect to the volume of cord blood, for example.
[0182] A method for preparing cord blood components using the cord
blood component preparation device 6 of the present embodiment will
be described with reference to an illustrative example. Specific
treatment conditions can be set to be the same as those described
above, unless otherwise stated.
[0183] First, a puncture needle is attached to the connection part
9 of the tube 68c. The tip of the puncture needle is inserted to an
umbilical cord, and cord blood is introduced to the tube 68c. Then,
the cord blood is introduced to the first storage unit 10 and the
second storage unit 60 through the tubes 68a and 68b. The order of
introducing the cord blood is not particularly limited, and it may
be introduced to these storage units at the same time or
sequentially. In the latter case, the introduction of the cord
blood can be carried out as follows, for example. First, the cord
blood is introduced to the second storage unit 60 with the flow
path of the tube 68a connected to the first storage unit 10 being
blocked in the vicinity of the branch connector 19. Thereafter, the
flow path of the tube 68b connected to the second storage unit 60
is blocked in the vicinity of the branch connector 19 and then the
blocking of the tube 68a is released to allow the cord blood to be
introduced to the first storage unit 10. After the cord blood is
introduced to both the first storage unit 10 and the second storage
unit 60, the tubes 68a and 68b between these storage units may be
cut off and fused.
[0184] In the first storage unit 10, an anticoagulant may be
accommodated in advance, or it may be introduced to the first
storage unit 10 prior to the introduction of cord blood, as
described above, for example. After cord blood is introduced to the
first storage unit 10 provided with the anticoagulant, it is
possible to obtain the above-described hematopoietic stem cells,
treated serum, and untreated serum by conducting the same
treatments as in the first embodiment, for example. The respective
accommodation units connected to the first storage unit 10 may be
the same as those in the second or third embodiment, instead of
those in the first embodiment, for example.
[0185] On the other hand, in the second storage unit 60, the blood
coagulation-accelerating substance may be accommodated in advance,
or it may be introduced to the second storage unit 60 prior to the
introduction of cord blood, as described above, for example. When
the cord blood is introduced to the second storage unit 60 provided
with the blood coagulation-accelerating substance, blood
coagulation (fibrin formation) occurs in the cord blood, and also,
platelets in the cord blood are activated. By this activation, more
growth factors are released as compared with the case where the
platelets are not activated. Then, after the blood coagulation has
occurred, the second storage unit 60 is centrifuged, thus achieving
the separation into a sediment fraction containing fibrin,
platelets, and the like and a supernatant fraction. This
supernatant fraction is introduced to the third serum accommodation
unit 61b through the tube 68d connecting the second storage unit 60
and the third serum accommodation unit 61b. Thus, serum containing
more growth factors can be collected. Furthermore, according to the
present embodiment, since the second storage unit 60 does not
contain an anticoagulant, serum free of a neutralizer such as
calcium chloride can be obtained, for example.
[0186] Next, examples of the present invention will be described.
It is to be noted, however, the present invention is by no means
limited by the following examples.
EXAMPLES
Example 1
[0187] Serum was prepared from cord blood, and the effect of the
serum on the proliferation and differentiation of cord blood
hematopoietic stem cells was examined.
[0188] (1) Preparation of Hematopoietic Stem Cells and Serum
[0189] 80 ml of cord blood containing a CPD solution as an
anticoagulant was introduced to a centrifuge tube, and HES (Hydroxy
Ethyl Starch) was added thereto as an erythrocyte sedimenting agent
so that its concentration became 12 mg/ml. The mixture was allowed
to stand still at room temperature for 60 minutes, and only a
supernatant fraction was collected. This was centrifuged (3920
m/s.sup.2 (400.times.g), 5 minutes), and a supernatant fraction and
a sediment fraction were obtained. The supernatant fraction is a
platelet-rich plasma fraction, and the sediment fraction is a
nucleated cell fraction.
[0190] 30 ml of the platelet-rich plasma fraction was introduced to
a centrifuge tube with a capacity of 50 ml. This was centrifuged
(22834 m/s.sup.2 (2330.times.g), 4.degree. C., 10 minutes), and a
supernatant fraction was obtained. The supernatant fraction was
heated at 56.degree. C. for 30 minutes, and then centrifuged (22834
m/s.sup.2 (2330.times.g), 4.degree. C., 10 minutes) so as to remove
fibrin therefrom. Thus, non-sonicated serum was prepared.
Hereinafter, this is referred to as cord blood serum (-). On the
other hand, 30 ml of the platelet-rich plasma fraction was
introduced to a PVC bag of 5 cm.times.10 cm and subjected to
sonication. Thereafter, a heat treatment and centrifuging were
performed under the same conditions as in the above. Thus,
sonicated serum was prepared. Hereinafter, this is referred to as
cord blood serum (+). The sonication was carried out for 30 minutes
using an ordinary ultrasonic generator under the conditions where,
when ultrasonic waves against an object to be treated were measured
with a sound pressure meter, a sound pressure of at least 5 mV
could be obtained, with the frequency being set to 45 kHz and the
distance from the ultrasonic generator being set to 15 cm.
[0191] The following treatment was performed with respect to the
nucleated cell fraction to purify hematopoietic stem cells. First,
to 0.3 ml of the nucleated cell fraction, 50 .mu.l of magnetic
beads (Miltenyi Biotec) having a CD34 antibody immobilized thereon
were added, and the resultant mixture was allowed to stand still at
4.degree. C. for 30 minutes. Then, this was applied to a magnetic
column (trade name "MS column", Miltenyi Biotec), and hematopoietic
stem cells were collected from the nucleated cell fraction. The
purity of the collected hematopoietic stem cells was found to be
about 70% through the examination by flow cytometry using a
fluorescent dye-labeled CD34 antibody (Beckman Coulter, Inc.).
[0192] The cord blood serum (+) and the cord blood serum (-) were
prepared from cord blood of each of three subjects (A, B, C), and
the cord blood hematopoietic stem cells were prepared from one (A)
of the three subjects.
[0193] (2) Culture of Hematopoietic Stem Cells
[0194] A medium having the following composition was prepared using
the cord blood serum (-) or the cord blood serum (+) of each of the
subjects A, B, and C as serum. Then, the hematopoietic stem cells
collected from the subject B were inoculated into each serum-added
medium at a density of 1.times.10.sup.4 cells/well and were
cultured in the medium at 37.degree. C. for 7 days.
TABLE-US-00001 TABLE 1 (Composition of Medium) Components
Concentration Iscove's modified Dulbecco's medium 90 v/v % (IMDM,
Gibco) Stem Cell Factor (PeproTech) 50 ng/ml Flt-3 ligand
(PeproTech) 20 ng/ml Thrombopoietin (R&D Systems) 10 ng/ml
Serum 10 v/v %
[0195] (3) Measurement of the Number of Cells
[0196] After the culture, the collected cells were analyzed by flow
cytometry using the above-described labeled CD34 antibody, and the
total number of cells per well, the number of hematopoietic stem
cells per well, and the proportion of the hematopoietic stem cells
to all the cells were calculated.
Comparative Example 1
[0197] Culture of hematopoietic stem cells and calculation of the
numbers of the respective cells were carried out in the same manner
as in Example 1, except that, in the culture of the hematopoietic
stem cells, fetal bovine serum (FBS) was used as serum.
Comparative Example 2
[0198] Culture of hematopoietic stem cells and calculation of the
numbers of the respective cells were carried out in the same manner
as in Example 1, except that, in the culture of the hematopoietic
stem cells, peripheral blood serum was used as serum. The
peripheral blood serum was prepared in the following manner. First,
20 ml of peripheral blood of an adult male was introduced to a
centrifuge tube containing two glass beads (bead diameter: 4 mm,
Bright Hyoshiki Kogyo K. K.). The centrifuge tube was shaken at
room temperature for 20 minutes, then centrifuged (22050 m/s.sup.2,
4.degree. C., 10 minutes), and the supernatant was collected. The
supernatant is a plasma fraction. This supernatant was heat-treated
at 56.degree. C. for 30 minutes, and filtered through a filter with
a pore size of 0.22 .mu.m (PVDF membrane, Millipore Corporation).
Thus, peripheral blood serum was prepared.
[0199] The graph of FIG. 7 shows the measurement results as to the
total number of cells and the number of hematopoietic stem cells
per well obtained in Example 1 and Comparative Examples 1 and 2. In
the graph of FIG. 7, the vertical axis indicates the number of
cells (.times.10.sup.4) per well after the culture, and the
respective bars indicate, from the left: the result obtained in
Comparative Example 1 in which FBS was used; the result obtained in
Comparative Example 2 in which the peripheral blood serum was used;
the results obtained in Example 1 when the cord blood serums (-) of
the subjects A to C were used; and the results obtained in Example
1 when the cord blood serums (+) of the subjects A to C were used.
The entire bar indicates the total number of cells, and the black
portion of the bar indicates the number of hematopoietic stem
cells.
[0200] As shown in FIG. 7, when the cord blood serums (-) were used
in Example 1, the proliferation of the hematopoietic stem cells was
observed as in Comparative Example 1 in which FBS was used and
Comparative Example 2 in which the peripheral blood serum was used.
From this result, it can be said that the cord blood serum (-) can
accelerate the proliferation of cord blood hematopoietic stem
cells. In contrast, when the cord blood serum (+) was used in
Example 1, the total number of the cells was much smaller than
those in Comparative Examples 1 and 2. Specifically, when the cord
blood serum (+) was used in Example 1, the total number of the
cells was 0.165.times.10.sup.4 to 1.0.times.10.sup.4 and inhibited
to 2.4% to 21.4% of the total number of the cells,
7.times.10.sup.4, in Comparative Example 1, and to 1.9% to 17.6% of
the total number of the cells, 8.5.times.10.sup.4, in Comparative
Example 2. From this result, it can be said that the cord blood
serum (+) can inhibit the proliferation of cord blood hematopoietic
stem cells. Further, when the cord blood serum (+) was used in
Example 1, the proportion of the hematopoietic stem cells to the
total number of the cells was 85% to 88%, which indicates that the
proportion of undifferentiated cells to the total number of the
cells was high. From this result, it can be said that the cord
blood serum (+) also can inhibit the differentiation of
hematopoietic stem cells. These results demonstrate that, since the
cord blood serum (+) can inhibit the proliferation and
differentiation and the cord blood serum (-) can accelerate the
proliferation, it is possible to control the proliferation of
hematopoietic stem cells as desired by combining them
appropriately. In particular, since hematopoietic stem cells to be
proliferated and cord blood serums (+) and (-) that can serve as
controlling agents can be prepared from the same cord blood, the
present invention provides excellent compatibility and safety and
also allows effective utilization of cord blood, which used to be
discarded. When the cord blood serum (+) was used in the culture of
mesenchymal stem cells, it was found that the proliferation was
accelerated, contrary to the above result. From this result, it is
interpreted that cord blood serum (+) can inhibit the proliferation
and differentiation of cord blood stem cells specifically.
Example 2
[0201] Hematopoietic stem cells, cord blood serum (+), and cord
blood serum (-) were prepared from cord blood of a single subject
in the same manner as in Example 1. Then, into the following media
to which the respective serums were added, the hematopoietic stem
cells were inoculated at a density of 5.times.10.sup.3 cells/well,
and cultured therein at 37.degree. C. for 14 days. The
concentration of the cord blood serum (+) in the medium was set to
5 and 10 v/v %, and the concentration of the cord blood serum (-)
in the medium was set to 0.125, 0.25, 0.5, 1, and 2.5 v/v %.
Furthermore, as a control, the hematopoietic stem cells were
cultured in the same manner using a medium containing neither of
the serums. Then, the cells collected after the culture were
analyzed by flow cytometry and the number of the hematopoietic stem
cells per well were calculated in the same manner as in Example
1.
TABLE-US-00002 TABLE 2 Components Concentration X-VIVO 10 (Takara
Bio Inc.) Predetermined Concentration (v/v %)* Stem Cell Factor
(PeproTech) 50 ng/ml Flt-3 ligand (PeproTech) 20 ng/ml
Thrombopoietin (R&D Systems) 10 ng/ml IL-6 (PeproTech) 50 ng/ml
sIL-6.alpha. (PeproTech) 50 ng/ml Serum Predetermined Concentration
(v/v %) *100 - serum concentration (v/v %)
[0202] The increase rate of the hematopoietic stem cells when the
media containing the cord blood serum (+) or the cord blood serum
(-) was used is shown in the table below. The increase rate was
determined as a relative value, assuming that the number of the
hematopoietic stem cells obtained after the culture of 14 days in
the serum-free medium was "100%".
TABLE-US-00003 TABLE 3 Concentration (v/v %) Increase rate (%) No
serum 0 100 Cord blood serum (+) 5 12 10 5 Cord blood serum (-)
0.125 128 0.25 156 0.5 156 1 121 2.5 124
[0203] As shown in Table 3, the increase rate of the stem cells
could be inhibited by culturing them in the medium containing the
sonicated cord blood serum (+) in the range from 5 to 10 v/v %. On
the other hand, the increase rate of the stem cells could be
increased by culturing them in the medium containing the
non-sonicated cord blood serum (-) in the range from 0.125 to 2.5
v/v %.
Example 3
[0204] Hematopoietic stem cells, cord blood serum (+), and cord
blood serum (-) were prepared from cord blood of a single subject
in the same manner as in Example 1. Then, into a medium containing
the cord blood serum (-) at a concentration of 0.5 v/v % as in
Example 2, the hematopoietic stem cells were inoculated at a
density of 5.times.10.sup.3 cells/well, and cultured therein at
37.degree. C. for 14 days. The medium containing the cord blood
serum (-) at a concentration of 0.5 v/v % was replaced with a
medium containing the cord blood serum (+) at a concentration of 10
v/v % as in Example 2 at the 14th day from the start of the
culture, and the stem cells were cultured further. Then, the cells
collected after the culture for predetermined culture periods (0,
7, 14, and 21 days) were analyzed by flow cytometry and the number
of hematopoietic stem cells per well were calculated in the same
manner as in Example 1. As a control, the culture was performed
using only the serum used initially from the start of the culture
under the same serum concentration condition without performing the
replacement with the medium containing the cord blood serum (+) at
a concentration of 10 v/v % at the 14th day of the culture, and the
number of hematopoietic stem cells were calculated.
[0205] The increase rate of the hematopoietic stem cells is shown
in the following table. The increase rate was determined as a
relative value (-fold), assuming that the number of hematopoietic
stem cells at the start of the culture (Day 0) was "1".
TABLE-US-00004 TABLE 4 With medium replacement Without medium
replacement Increase rate Increase rate Day 0 1-fold 1-fold Day 7
3.7-fold 3.9-fold Day 14 12.7-fold 11.0-fold Day 21 10.1-fold
30.2-fold
[0206] As can be seen from Table 4, by culturing the hematopoietic
stem cells in the medium containing the cord blood serum (-) at a
concentration of 0.5 v/v % and then replacing the medium with the
medium containing the cord blood serum (+) at a concentration of 10
v/v % on the 14th day of the culture, the increase in the
hematopoietic stem cells was inhibited.
INDUSTRIAL APPLICABILITY
[0207] As specifically described above, according to the present
invention, it is possible to control the proliferation and
differentiation of cord blood hematopoietic stem cells using serum
derived from cord blood without using a virus vector having a
problem in safety as described above, for example. Specifically,
the above-described control is possible using only cord blood
serum. Therefore, the present invention is very excellent in safety
and can regulate the proliferation and differentiation with simple
operations. In particular, since the present invention uses serum
derived from cord blood, it is possible to use hematopoietic stem
cells and serum derived from cord blood of the same individual, for
example. Therefore, cord blood can be used more effectively, and
since the components derived from the same individual can be used
in combination, the reliability regarding the safety also can be
improved. As described above, since the present invention can
inhibit the proliferation and differentiation of hematopoietic stem
cells, it is particularly useful when delivering the hematopoietic
stem cells to a destination, storing the hematopoietic stem cells
until a desired time at which the proliferation of the
hematopoietic stem cells is started, and the like, for example.
Thus, it can be said that the method according to the present
invention can further promote the effective utilization of cord
blood hematopoietic stem cells in the field of regenerative
medicine.
* * * * *