Chemistry: molecular biology and microbiology – Apparatus – Differentiated tissue perfusion or preservation apparatus
Reexamination Certificate
2000-12-15
2002-09-10
Redding, David A. (Department: 1744)
Chemistry: molecular biology and microbiology
Apparatus
Differentiated tissue perfusion or preservation apparatus
C435S288700, C435S297500
Reexamination Certificate
active
06448063
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to an experimental apparatus for a physiological experiment of a sliced specimen (slice preparation) of biological tissues while maintaining the physiological activities of the sliced specimen of the biological tissue, and a specimen holder (slice holder) to be used in the experimental apparatus.
2. Description of the Related Art
It is a basic technique of the physiological experiments to excise a part of the tissue from a biological specimen and measure its physiological activities while maintaining its physiological activities. Mostly, a sliced specimen excised from the biological tissue (simply abbreviated as a sliced specimen hereinafter), or a thin tissue piece (usually with a thickness of 1 mm or less) excised from the biological specimen is prepared:, and various physiological measurements are carried out using the specimen, because it is necessary to feed oxygen and nutrients over the entire biological tissue and remove waste products in order to maintain physiological activities of the biological tissue.
Two methods have been used for storing the sliced specimen for a long period of time while maintaining its physiological activities. The first method comprises, as shown in
FIG. 5
, disposing a petri dish
12
with a mesh structure for allowing a solvent to permeate in a vessel
11
filled with a saline solution
1
containing appropriate nutrients, placing a:sliced specimen
13
on the petri dish
12
, and immersing and maintaining the sliced specimen
13
in the saline solution
1
while a gas mainly comprising oxygen and carbon dioxide is continuously fed into the saline solution
1
through a gas feed tube
15
equipped with a spherical filter
14
.
The second method comprises, as shown in
FIG. 6
, placing a filter
17
(filter paper or membrane filter) that is permeable to both a solution or gas on a vessel
16
filled with the saline solution
1
, mounting a sliced specimen
13
on the filter, housing the overall vessel
16
in a sealed vessel
18
, and filling the inside of the sealed vessel
18
with a moist gas generated from the moisturizing saline solution
1
.
The apparatuses shown in
FIGS. 5 and 6
may be also used for staining the biological specimen with a biological dye. In the apparatus shown in
FIG. 5
, the sliced specimen
13
is stained by adding a dye in the solution
1
in which the specimen is immersed. In the apparatus shown in
FIG. 6
, on the other hand, the sliced specimen
13
is stained by dropping a staining solution on the specimen.
While both of the methods described above have almost the same effect for maintaining the sliced specimen
13
for a long period of time, it is a problem that the physiological activity of the sliced specimen
13
has been decreased because the sliced specimen
13
tends to be deformed and damaged when the sliced specimen
13
is transferred to a different site by taking the specimen out of the vessels
11
and
18
.
While the sliced specimen
13
is transferred by sucking it with a pipette together with the solution
1
in the first method, deformation of the thin and soft sliced specimen
13
is inevitable, sometimes dealing a fatal damage to the sliced specimen
13
.
While the sliced specimen
13
is peeled off from the filter
17
to transfer it into the solution in a separate vessel, the sliced specimen
13
is also liable to be imparted with a fatal damage during the transfer process. Since the sliced specimen
13
stored for a long period of time is often adhered onto the filter
17
, a special care is required for peeling the sliced specimen
13
from the filter
17
.
A high concentration of the dye should be maintained for a long period of time around the sliced specimen
13
in order to sufficiently and uniformly stain the biological specimen. Accordingly, the dye should be distributed in the overall solution
1
dipping the sliced specimen
13
when the apparatus shown in
FIG. 5
is used for staining, which is very uneconomical because a large quantity of the usually expensive dye is needed. While consumption of the dye may be saved in the apparatus shown in
FIG. 6
, staining tends to be poor since it is difficult to allow a sufficient amount of the staining solution to stay around the sliced specimen
13
.
Properly introducing the sliced specimen
13
into a measuring apparatus And holding the specimen there is important for measuring the physiological activities of the sliced specimen
13
. The sliced specimen
13
is introduced into the measuring apparatus using a pipette together with the solution, as has been described previously. However, the physiological activities of the sliced specimen
13
may be possibly compromised by the impact of the transfer operation or by environmental changes.
It is also necessary to immerse the sliced specimen
13
in the saline solution
1
, and the solution around the sliced specimen
13
should be steadily circulated in the measuring apparatus. While an appropriate weight is used for fixing the sliced specimen
13
in order to avoid the sliced specimen
13
from being displaced by the stream of the solution, the physiological activities of the sliced specimen
13
may be compromised by stress caused by the weight. The weight may also interfere the physiological measurements such as observation of transmission light and electrical potential measurements, and imaging using a dye.
The apparatus shown in
FIG. 6
may be used for a cultivator by maintaining the inside of the sealed vessel
18
aseptic. However, it is difficult for the sliced specimen
13
to be transferred into the measuring apparatus without being damaged after peeling off the sliced specimen
13
from the filter
17
, because the sliced specimen
13
is adhered on the filter
17
. It is also impossible in general to withdraw the sliced specimen
13
, subject to a aseptic treatment and resume culturing by putting it back to the cultivator after the measurement without dealing a damage to the specimen.
SUMMARY OF THE INVENTION
Accordingly, it is an object of the present invention devised for solving the technical drawbacks of the conventional art to provide an experimental apparatus for a sliced specimen of biological tissues and a specimen holder, wherein the conditions required for preservation, staining, physiological measurements and cultivation are satisfied while maintaining the sliced specimen in a condition suitable for maintaining physiological activities in each conditions above, besides subjecting the sliced specimen to an experiment by readily permitting the specimen to be transferred without being physically damaged.
A first aspect of the present invention provides an experimental apparatus for a sliced specimen of a biological tissue that is used for a physiological experiment by holding the sliced specimen of the biological tissue in a circulating saline solution while maintaining its physiological activities, comprising an experimental vessel for housing the saline solution, a specimen holder for holding the sliced specimen of the biological tissue, and a device for holding the holder for allowing the specimen holder to be attached to and detached from a prescribed position in the experimental vessel.
A second aspect of the present invention provides the specimen holder comprises an annular holder frame and a membrane filter for closing the lower part of the holder frame, the sliced specimen of the biological tissue being held on the membrane filter, in the experimental apparatus for a sliced specimen of a biological tissue of the first aspect.
A third aspect of the present invention provides holder frame has a height by ⅕ or less as small as its inner diameter, in the experimental apparatus for a sliced specimen of a biological tissue of the second aspect.
A fourth aspect of the present invention provides the membrane filter is made of a material on which the sliced specimen of the biological tissue can adheres, in the experimental apparatus for a sliced specimen of a biological tissu
Ichikawa Michinori
Tominaga Takashi
Redding David A.
Riken
Rosenthal & Osha L.L.P.
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