JP5120998
TISSUE-PRESERVING SOLUTION
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a tissue preservation
solution that is useful in the field of medicine, medical
experiment, etc.
2. Description of the Related Art
In the medicine that is making rapid advances in recent
years, the importance of tissue (as well as organ and cell,
hereinafter the same shall apply) transplant increases, and the
advances are supported by the progress, for example, in
immunosuppressive agents and tissue preservation technology. In
the tissue transplant, it is ideal that the tissue removed from a
donor be transplanted immediately to a recipient, but it is not
always transplanted immediately. Thus, it is extremely important
how the removed tissue is to be preserved, and currently a better
tissue preservation solution has been desired.
In recent years, attention has been paid to various bioactive
effects of water that contains nanobubbles of oxygen in large
amount (oxygen-nanobubble water) on living organisms. For example,
oxygen-nanobubble water improves adaptability of fish and
shellfish to environmental change, or restores a debilitated
individual quickly (See, for example, Japanese Patent Application
Laid-Open UP-A) No. 2005-246294). Nanobubbles are ultrafine
bubbles with a diameter of 1 µm or less, and are typically
generated in the process where microbubbles (minute bubble with a
diameter of 50 µm or less) shrink. Since nanobubbles are
self-pressurized by the action of surface tension, they are
completely dissolved rapidly. Thus, the lifetime was considered to
be short in general. However, it is reported that in the case
where nanobubbles are coated with shell by a surfactant, or in the
case where they are subjected to electrostatic repulsion due to
surface charging, even bubbles in nano-order can exist for a
certain period. Especially, nanobubbles stabilized due to charging
effect retain properties as bubble; thus various applications,
such as direct action to organisms at cellular level, are expected
(See, for example, JP-A No. 2005-246294).
BRIEF SUMMARY OF THE INVENTION
An object of the invention is to solve the conventional
problems and to achieve the following objects. Specifically, an
object of the invention is to provide a tissue preservation
solution that has excellent tissue-preserving ability and is
useful in the field of medicine, medical experiment, etc.
In order to solve the problems, inventors of the present invention
have investigated vigorously and have found the following
experiences or discoveries. Specifically, they have found that
oxygen-nanobubble water can exhibit tissue-preserving effect
similar to, or more than the conventional tissue culture mediums
such as Dulbecco's modified Eagle medium (DMEM) and neuron culture
medium (NCM).
As described above, it has been known that oxygen-nanobubble water
has various bioactive effects on living organisms. For example, it
improves adaptability of fish and shellfish to environmental
change, or restores a debilitated individual quickly. Thus, the
oxygen-nanobubble water is attracting attention. In addition,
recently a method for producing oxygen-nanobubble water has been
established that can maintain nanobubbles stably for a long time
(See, for example, JP-A No. 2005-246294).
Previously, however, it has not been known that the
oxygen-nanobubble water can exhibit tissue-preserving effect
similar to, or more than the conventional tissue culture mediums,
and that therefore, the oxygen-nanobubble water can be suitably
utilized as an excellent tissue preservation solution in the field
of medicine, medical experiment, etc., which was newly found by
the inventors of the present invention.
Accordingly, the invention is based on the above-mentioned
findings by the inventors of the present invention. The means for
solving the problems is a tissue preservation solution including
oxygen nanobubbles.
The invention can solve the conventional problems and can provide
a tissue preservation solution that has excellent
tissue-preserving ability and is useful in the field of medicine,
medical experiment, etc.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
FIG. 1 is a schematic view showing each part of rat vagus
nerve tissue and each preservation method that were used in
Example 1.
FIG. 2 is optical microscope images (H.E. staining: ×400) of a
normal control vagus nerve tissue (left side: L1).
FIG. 3 is optical microscope images (H.E. staining: ×400) of
each vagus nerve tissue (left side: parts represented by A).
FIG. 4 is optical microscope images (H.E. staining: ×400) of
each vagus nerve tissue (right side: parts represented by B).
DETAILED DESCRIPTION OF THE INVENTION
(Tissue Preservation Solution)
The tissue preservation solution of the invention
comprises oxygen nanobubbles.
<Oxygen-Nanobubble Water>
In the invention, “oxygen nanobubble” refers to a bubble
which comprises oxygen and which has a nano-order diameter (1 µm
or less). “Oxygen-nanobubble water” refers to an aqueous solution
that comprises oxygen wherein the oxygen exists as the oxygen
nanobubble. Specifically, the oxygen-nanobubble water is a
preferable aspect of the tissue preservation solution of the
invention.
Oxygen nanobubbles present in oxygen-nanobubble water for use in
the tissue preservation solution of the invention may have a
variety of bubble diameters. Among them, the bubble diameter is
preferably 200 nm or less, more preferably 100 nm or less and most
preferably less than 10 nm. Oxygen nanobubbles with a diameter of
100 nm or less are advantageous in that there is less possibility
for incorporation of electrolytes or foreign substances. Oxygen
nanobubbles with a diameter less than 10 nm are further
advantageous in that incorporation of many foreign substances
including viruses can be prevented.
The bubble diameter of the oxygen nanobubble can be adjusted to a
desired size using, for example, a reverse osmosis membrane. The
bubble diameter of the oxygen nanobubble can be measured with, for
example, a dynamic light scattering equipment.
The oxygen concentration of the oxygen-nanobubble water is
preferably saturated level. It is important that the oxygen
nanobubbles be dissolved in an aqueous solution sufficiently.
The salt concentration, pH, and hardness of the oxygen-nanobubble
water are not particularly limited and can be appropriately
selected depending on the application. For example, each of them
can be adjusted to a desired degree during the production process
of oxygen-nanobubble water described below or after the production
of oxygen-nanobubble water.
—Other Element—
The oxygen-nanobubble water may comprise other elements
than the oxygen nanobubbles as necessary. Specific examples of the
other element are not particularly limited and can be
appropriately selected depending on the application, including,
for example, iron, manganese, and salts.
—Production—
The oxygen-nanobubble water can be produced by any method
without limitation and the method can be appropriately selected
depending on the application. For example, the oxygen-nanobubble
water can be produced by the production method disclosed in JP-A
No. 2005-246294. The production method disclosed in the gazette is
preferable since it can produce oxygen-nanobubble water in which
oxygen nanobubbles exist stably and do not disappear from the
aqueous solution over a long period of several months or more.
The salt concentration of the aqueous solution used in the
production process of the oxygen-nanobubble water is preferably
0.2% by mass to 3.0% by mass, and more preferably 0.8% by mass to
1.2% by mass. When the salt concentration is within the range of
from 0.8% by mass to 1.2% by mass, nanobubbles (core of gas) can
be produced easily; thus it is advantageous in that production
efficiency of the oxygen-nanobubble water is excellent. The salt
concentration can be measured using, for example, a known
instrument for measuring salt concentration.
It is considered that the pH and hardness of the aqueous solution
used in the production process of the oxygen-nanobubble water does
not affect the production efficiency of oxygen nanobubbles as
greatly as does the salt concentration. Typically, the pH is
preferably 7 to 8, and the hardness is preferably 20 to 30. The pH
and hardness can be measured using, for example, a known
instrument for measuring pH and a known instrument for measuring
hardness, respectively.
In the production process of the oxygen-nanobubble water, iron,
manganese, and/or salts can be preferably added.
Specifically, oxygen microbubbles of 50 µm or less are produced
using hard water (groundwater) with a salt concentration of 1.0%
by mass as a raw material. Then, by rapidly collapsing or crushing
the oxygen microbubbles, oxygen-nanobubble water can be produced.
Further, purified oxygen-nanobubble water (salt content: 0% by
mass), “Naga no shizuku” (drips of Naga) (manufactured by NAGA
Co., Ltd.), can be prepared by passing the resulting
oxygen-nanobubble water through a reverse osmosis membrane of 10
nm. “Naga no shizuku” is drinking water approved by the Ministry
of Health, Labour and Welfare. Meanwhile, oxygen-nanobubble water
with a salt concentration of 1.0% by mass is oxygen-nanobubble
water that is not passed through a reverse osmosis membrane of 10
nm. Varying the mixing ratio of both of the oxygen-nanobubble
waters can provide oxygen-nanobubble waters with a salt
concentration of from 0% by mass to 1.0% by mass.
The oxygen-nanobubble water prepared as mentioned above may be
used as the tissue preservation solution itself, however, for
example, the oxygen-nanobubble water may be added to an existing
tissue preservation solution, or a tissue preservation solution
that includes the oxygen-nanobubble water as an ingredient and has
a conventional composition may be prepared. This will further
enhance capability to preserve tissues. Since oxygen-nanobubble
water is aseptic water and has a high disinfecting ability, it is
suitably used for preparing tissue preservation solution by means
of such methods. Thus, such tissue preservation solutions as
mentioned above that utilize oxygen-nanobubble water in part are
also included within the scope of the tissue preservation solution
of the invention.
<Tissue>
The object to be preserved in the tissue preservation
solution not particularly limited and can be appropriately
selected depending on the application. The tissue preservation
solution of the invention is also suitable for the preservation of
“cell” or “organ”.
The “tissue” is not particularly limited and can be appropriately
selected depending on the application; examples thereof include
epithelial tissues, connective tissues, muscular tissues, and
nerve tissues.
The “cell” is not particularly limited and can be appropriately
selected depending on the application; examples thereof include
epidermal cells, pancreatic parenchymal cells, pancreatic ductal
cells, hepatic cells, blood cells, cardiac muscle cells, skeletal
muscle cells, osteoblasts, skeletal myoblasts, nerve cells,
endothelial cells, pigment cells, smooth muscle cells, fat cells,
bone cells, and cartilage cells.
The “organ” is not particularly limited and can be appropriately
selected depending on the application; examples thereof include
skin, blood vessel, cornea, kidney, heart, liver, umbilical cord,
intestine, nerve, lung, placenta, pancreas, brain, peripheral
extremities, and retina.
The organism from which the tissue, cell, and organ is derived is
not particularly limited and can be appropriately selected
depending on the application. The tissues, cells, and organs
derived from a mammal are preferable, and tissues, cells, and
organs derived from a human are more preferable.
<Use>
The tissue preservation solution may be used without
limitation. The tissue preservation solution can be used in the
same manner as a conventional tissue preservation solution. For
example, the tissue, cell, organ, or the like is removed from a
living body, and then it is immersed in the tissue preservation
solution for a desired period.
The tissue preservation solution itself can be stored by any
method without limitation and the method can be appropriately
selected depending on the application.
Since the tissue preservation solution has excellent
tissue-preserving ability, it can be suitably utilized, for
example, as a tissue preservation solution in the field of
medicine, medical experiment, etc.
EXAMPLES
Examples of the invention are illustrated below, but
these are not to be construed as limiting the invention.
Example 1
Evaluation of Effect of Oxygen-Nanobubble Water on Tissue
Preservation
Oxygen-nanobubble water (REO Laboratory Co., Ltd.), one
aspect of the tissue preservation solution of the invention, was
prepared with reference to the production method disclosed in JP-A
No. 2005-246294. Specifically, oxygen microbubbles of 50 µm or
less was produced using hard water (groundwater) with a salt
concentration of 1.0% by mass as a raw material. Then, by rapidly
collapsing or crushing the oxygen microbubbles, oxygen-nanobubble
water was produced.
The effect of the prepared oxygen-nanobubble water on tissue
preservation was histologically investigated using rat vagus nerve
as a material. Physiological saline and conventional tissue
culture medium were used as a control.
<Method>
—Storage/Fixation of Tissue—
Four rats (rats 1 to 4) (Std: Wistar/ST, 10 weeks of age,
male, 300 g) were anesthetized with Nembutal, and vagus nerves on
both sides (total 8 nerves) were removed using an operation
microscope. Next, of the removed vagus nerves, vagus nerves (total
two nerves) removed from one rat (rat 1) were used for the
following experimental operations without cutting. The vagus
nerves (total six nerves), removed from the remaining three rats
(rats 2 to 4), were each cut into five pieces.
Immediately after the cutting or removal, the part very near to
the central nerve, (L2 to L4, R2 to R4), of each vagus nerve
removed from three rats (rats 2 to 4) and the entire vagus nerve
removed from the remaining one rat (rat 1), as a normal control
vagus nerve tissue, were fixed with Zamboni's fixative (15% picric
acid, 4% paraformaldehyde, 0.4 M phosphate buffer, pH 7.4) at 4°
C. for one week. Also, the rest of each vagus nerve tissue removed
from three rats (rats 2 to 4) was divided and each part of the
tissue was stored, starting from the part near to the central
nerve, in physiological saline (C), oxygen-nanobubble water (NB),
Dulbecco's modified Eagle medium (DMEM), and neuron culture medium
(NCM), respectively, at 4° C. (FIG. 1, where left side and right
side are represented by A and B, respectively). After 1, 3, or 7
days, each part of the tissue was placed in Zamboni's fixative and
fixed at 4° C. for one week. Further, each fixed vagus nerve
tissue was embedded using a low melting point-paraffin (52° C.).
Each part of the rat vagus nerve tissue shown in FIG. 1
corresponds to each storage method as follows.
[Storage Method]
L1 to L4, R1 to R4: fixed immediately after the cutting
or removal
C-A1, 3, 7, C-B1, 3, 7: stored in physiological saline
NB-A1, 3, 7, NB-B1, 3, 7: stored in oxygen-nanobubble water
DMEM-A1, 3, 7, DMEM-B1, 3, 7: stored in DMEM
NCM-A1, 3, 7, NCM-B1, 3, 7: stored in NCM
[Period of Storage]
A1, B1: stored for 1 day
A3, B3: stored for 3 days
A7, B7: stored for 7 days
—Hematoxylin-Eosin Staining (H. E. Staining)—
Each paraffin-embedded vagus nerve tissue was sliced at 8 µm using
a microtome, and then placed on MAS-coated glass slide. Sections
were deparaffinized through a xylene series and an alcohol series,
and alcohol was removed from deparaffinized sections by rinsing in
distilled water for 5 minutes. Then, the nuclei were stained at
room temperature for 30 minutes using Mayer's hematoxylin
solution. Further, after washing in running water for 15 minutes
to blue the nuclei, fiber, stroma, and the like were stained with
10% eosin solution for 5 minutes. Sections were washed in running
water for 1 minute, followed by distilled water. Then, excess
eosin was removed and sections were dehydrated with an alcohol
series, cleared through a xylene series, and then mounted with
Entellan neu to prepare H. E. stained tissue specimens (See, for
example, Reference Literatures (1) and (2) below).
Next, the prepared H. E. stained tissue specimens were examined
using an optical microscope, and the state of the tissue after the
storage was evaluated histologically by examining microscopic
morphological changes of each vagus nerve tissue (See, for
example, Reference Literatures (3) to (8)).
<Results>
Results are shown in FIGS. 2 to 4 and Table 1.
For the microscopic morphology of the normal control vagus nerve
tissue, histological differences were not observed between central
side, center, and peripheral side (FIG. 2). Also, histological
differences were not observed between left side and right side,
and between individuals.
In the case of storage in physiological saline (C), there was a
significant thinning due to the cytoplasmic loss in the vagus
nerve tissue stored for 3 days or later. In the case of storage in
DMEM, a certain degree of cytoplasmic loss was observed in the
vagus nerve tissue stored for 3 days, and there was a significant
thinning due to the cytoplasmic loss in the vagus nerve tissue
stored for 7 days. In contrast, in the case of storage in
oxygen-nanobubble water (NB) or NCM, the tissue were preserved
relatively well, and only a mild degree of cytoplasmic loss was
observed in the vagus nerve tissue stored for 7 days (FIGS. 3 and
4).
For each vagus nerve tissue, the state of tissue after storage was
evaluated histologically according to the following evaluation
standards. The results are shown in Table 1.
[Evaluation Standards]
3+: Maintenance of good microscopic morphology
2+: Mild degree of cytoplasmic loss
+: Significant thinning due to the cytoplasmic loss
TABLE 1
Storage Storage Storage
for for 3 for 7
Storage condition 1 day days days
Left side Physiological saline 3+
+ +
(represented by A) Oxygen nanobubble 3+ 2+
2+
water
DMEM 3+ 2+ +
NCM 3+ 2+ 2+
Right side Physiological saline 2+ + +
(represented by B) Oxygen nanobubble 2+ 2+
2+
water
DMEM 2+ + +
NCM 2+ 2+ 2+
As shown in results of Table 1, in the case of the vagus nerve
tissue stored in oxygen-nanobubble water, there was less thinning
due to the cytoplasmic loss in the vagus nerve tissue and the
microscopic morphology of the vagus nerve tissue was maintained
better, compared to those stored in physiological saline and in
DMEM. In addition, the vagus nerve tissue stored in oxygen
nanobubble was preserved as well as that stored in NCM. The
tissue-preserving ability of each preservation solution used in
this Example 1 was in the order of oxygen-nanobubble
water?NCM>DMEM>physiological saline.
The above-mentioned results indicate that the tissue preservation
solution of the invention, which comprises oxygen nanobubbles, has
excellent tissue-preserving ability, suggesting that it can be
applied as a tissue preservation solution in the field of
medicine, medical experiment, etc.
[Reference Literature]
Reference literatures in the Examples are as follows.
(1) Shinkichi Akao, Akira Aso, Shinji Adachi, kenichi Anan,
Michiko Abe et al. (1997) “Shin senshokuho no subete” (all
staining methods), Ishiyaku Publishers, Inc., 3-6
(2) Moe Shimotori (2004) “Functional and histological study of
transection and regeneration pattern of vagus nerve” graduation
thesis, Tokyo Medical and Dental University
(3) Yoshifusa Shimizu, Chizuka Ide, Koki Kawamura, Shigeo Toya,
Hideo Togi (1997) “Regeneration and functional rebuilding of the
nerves—basic and clical medicine—”, Nishimura-Shoten, 227-249
(4) Bray G M, Aguayo A J (1974) “Regeneration of peripheral
unmyelinated nerves. Fate of the axonal sprouts which develop
after injury”, J Anat, 117:517-529
(5) Barbara Young, John W Heath (2001) “Functional Histology A
Text and Colour Atlas” 4th edition, Japanese version, Igaku-Shoin,
116-142
(6) Alan Stevens, James Lowe (1999) “Human Histology”, translation
of the second edition, Nankodo, 77-98
(7) Leslie P Gartner, James L Hiatt (2003) “Color Textbook of
Histology” Nishimura-Shoten, 157-187
(8) Leslie P Gartner, James L Hiatt (1999) “Color Atlas of
Histology”, Japanese version, Medical Sciences International,
126-146
The tissue preservation solution of the invention has good
tissue-preserving ability and is thus useful as a tissue
preservation solution in the field of medicine, medical
experiment, etc.
