JPH10165166A - Cell-fixing plate and method for fixing cell to cell-fixing plate - Google Patents

Abstract

PROBLEM TO BE SOLVED: To stably fix plural cells at specific positions of a cell-fixing plate. SOLUTION: Plural dent parts 3 each of which has a shape fitting to the lower side of a fertilized egg 11 are formed on the surface side of a porous plate 20 having fine pours 21, and a liquid-impermeable film 31 is formed on the region of the surface of the plate 20 other than the dent parts 3. When an egg floating liquid is supplied onto the surface side of such a cell-fixing plate 2, and suction is applied from the backside with a specified pressure, the fluid component of the egg floating liquid is repulsed in the region where the liquid-impermeable film is formed, and a flow of the fluid component toward the dent parts 3 is generated. Fertilized eggs 11 are sucked into the dent parts 3 by the liquid flow, sucked onto the whole of the inside surface of the dent parts 3 and fixed there in a stable state.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a plate for fixing cells used, for example, for microinjection and the like, and a method for fixing cells to the plate for fixing cells.

[0002]

2. Description of the Related Art In a microinjection method, a substance can be injected into a specific cell, and a gene is also injected into a fertilized egg by this method. Conventionally, when a gene is injected into a fertilized egg such as a mini-pig by this method, the fertilized egg has a very small diameter of about 150 μm. For example, as shown in FIG. 10, an operator observes the fertilized egg 11 with a binocular microscope. While a part of the nucleus 12 of the fertilized egg 11 was suction-fixed with the tip of the micropipette 13 on a flat liquid-impervious plate, the microneedle 14 was inserted into the fertilized egg 11 to generate the gene. Is performed by injecting.

[0003]

However, in the above-mentioned method, a part of the fertilized egg 11 is
, It is an unstable state even if it is fixed. Therefore, when trying to insert the micro needle 14 in this state, the egg nucleus is deformed at the time of insertion, making the insertion operation difficult or damaging the fertilized egg 11 at the time of insertion. There are cases. Also, since the binocular microscope can only see a flat surface, even if it is thought that the microneedle 14 has been inserted into the fertilized egg 11,
The microneedle 14 is located at a distance from the fertilized egg 1
In some cases, no gene is injected into one.

[0004] Therefore, the operation of injecting a gene into a fertilized egg by this method requires skill and experience of an operator, and generally the work efficiency is poor. In addition, since the operator performs operations using the binocular microscope, there is also a problem that both eyes are restrained and fatigue is increased, and the burden on the operator is increased.

The present invention has been made under such circumstances, and an object of the present invention is to provide a cell-fixing plate and a cell-fixing plate capable of fixing a plurality of cells at predetermined positions on a cell-fixing plate. Another object of the present invention is to provide a method for fixing cells to a plate for cell fixing, and another object is to provide a cell fixing plate capable of stably fixing a plurality of cells at a predetermined position on a plate for cell fixing. And a method for fixing cells to a plate for fixing cells.

[0006]

For this reason, the cell fixing plate of the present invention comprises a plurality of recesses for accommodating cells on one side of a plate made of a liquid-permeable material such as microporous glass. Is formed. Here, it is preferable that the recess is formed in a shape in which a part of the cell is fitted, and it is preferable that a liquid-impermeable membrane is formed in a region other than the recess on one surface side of the plate.

Further, the method of fixing cells to a cell fixing plate according to the present invention is directed to a cell fixing plate having a plurality of recesses for accommodating cells on one side of a plate made of a liquid-permeable material. In a method of fixing a cell in the recess by storing a plate in a storage chamber, supplying a liquid containing cells to one side, and sucking the liquid from the other side, the pressure in the storage chamber is detected. It is characterized in that it is determined whether or not cells have been fixed in the concave portion based on the detected value.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention is directed to a method in which a liquid supplied to the front side of a plate made of a liquid-permeable material, for example, a porous material having fine pores, is sucked from the back side thereof. It is made by paying attention to permeation toward the back side through the micropores of the porous plate. For example, a cell fixing plate used for microinjection and this cell fixing plate are used. This shows a preferred example of a method for fixing cells to a cell.

First, an example of an embodiment of the cell fixing plate of the present invention will be described. FIG. 1A is a plan view of a cell fixing plate 2, FIG. 1B is a cross-sectional view taken along a line AA, and FIG. 2 is an enlarged cross-sectional view of a main part. In FIG. 1, reference numeral 20 denotes a porous plate formed of, for example, a liquid-permeable material, such as porous glass, formed in a disk shape having a diameter of 8 mm and a thickness of 0.77 mm. Here, the liquid-permeable material is
A material that has the property that the liquid supplied to the front side permeates toward the back side, and the material where the liquid supplied to the front side permeates toward the back side when sucked from the back side Shall be included.

The porous plate has a large number of micropores 21 having a diameter of, for example, about 2 μm.
It has a porous structure of about 25 cc / g and is made of, for example, microporous glass. In such a porous plate 20, when sucked from the back side, the liquid supplied to the front side permeates toward the back side.

Here, the microporous glass is
This is a porous glass utilizing the phase separation phenomenon of glass, and is produced, for example, in the process of producing high silicate glass. Since the size of the ultrafine pore diameter is determined by the phase separation generated by the heat treatment, a microporous glass having micropores of a desired size can be manufactured by changing the heat treatment temperature and the heat treatment time.

On the surface of such a porous plate 20, for example, 25 concave portions 3 are formed. For example, as shown in FIG. 2, the concave portion 3 is formed so as to be compatible with a cell to be fixed, for example, a part of a fertilized egg 11 of a miniature pig. In this example, for example, a spherical fertilized egg having a diameter of about 150 to 180 μm. 11 is formed in a substantially hemispherical shape with an opening diameter of about 140 μm and a depth of about 70 μm so that the lower side of the projection 11 is fitted. The fertilized egg 11 is fixed in such a concave portion 3 such that the entire lower surface of the fertilized egg 11 fits into the inner surface of the concave portion 3 and the upper side of the fertilized egg 11 projects from the upper surface of the porous plate 20.

The recesses 3 are arranged substantially in the center of the porous plate 20 in, for example, 5 rows × 5 rows, and the interval between the recesses 3 adjacent in the vertical and horizontal directions is 250 μm.
Set to about. A liquid-impermeable film 31 made of a liquid-impermeable material such as glass is formed in a region other than the concave portion 3 on the surface of the porous plate 20. The liquid-impermeable membrane 31 prevents permeation of the liquid component of the egg suspension, for example. In a region where the liquid-impermeable membrane 31 is formed, liquid permeates into the porous plate 20. Is suppressed.

Such a cell fixing plate 2 is, for example, a porous plate 2 having micropores 21 of a desired size.
Is manufactured by coating the entire surface of the porous plate 20 with a liquid-impermeable material, for example, to form a liquid-impermeable film 31, and then forming the concave portion 3 by, for example, sandblasting. Is done.

Next, a method of fixing cells to the above-mentioned cell fixing plate 2 will be described. First, an example of an apparatus for carrying out this fixing method will be described with reference to FIG. 3. In FIG. 3, reference numeral 4 denotes a storage chamber for storing a plate 2 for fixing cells. The storage chamber 4 includes a bottomed tubular body 4A and a rod body 4B having a shape adapted to the inner surface of the bottomed tubular body 4A, and is configured by screwing the rod body 4B to the bottomed tubular body 4A. Have been.

The bottom surface of the bottomed cylindrical body 4A and the rod body 4B
Ring body 41 made of, for example, silicon
A cell fixing plate 2 sandwiching the upper and lower peripheral regions by a and 41b is provided, and the cell fixing plate 2 is stored in the storage chamber 4 in this manner. The ring bodies 41a and 41b are formed to have, for example, an outer diameter of 8 mm, a width of 2 mm, and a thickness of 0.5 mm, and are located outside a region where the concave portion 3 of the cell fixing plate 2 is formed. Is provided so as to cover the peripheral region, and acts as a seal so that the liquid component does not leak from between the outer peripheral edge of the cell fixing plate 2 and the inner surface of the storage chamber 4. In the storage chamber 4, for example, immediately below the cell fixing plate 2,
A pressure detecting means 42 composed of, for example, a mercury pressure gauge for detecting the pressure in the storage chamber 4 is provided.

The inner diameter a of the bottomed cylindrical body 4A is, for example, 9 m.
m, and approximately at the center of the upper surface of the bottomed cylindrical body 4A,
In order to supply the egg suspension to the plate 2 for cell fixation, an egg suspension supply port 43 having an inner diameter b of, for example, 3 mm is formed, and the rod body 4B has a back surface of the plate 2 for cell fixation. Suction path 4 with an inner diameter c of 3 mm for suctioning the side
4 are formed.

At the lower end of the suction passage 44 of the storage chamber 41, an exhaust means such as a vacuum pump 5 is provided through an exhaust pipe 51. An egg suspension tank 61 is provided downstream of the vacuum pump 5. Have been. On the other hand, an egg suspension supply tank 62 is provided on the upstream side of the storage chamber 4, and the egg suspension in this tank is fixed to the cells by the egg suspension supply pipe 63 through the supply port 43 of the storage chamber 4. It is configured to be supplied to the surface of the work plate 2. The egg suspension tank 61 and the egg suspension tank 62 are connected by a circulation supply pipe 64. In the figure, V1 and V2 denote valves and P denotes a pump, respectively.

In such an apparatus, when cells, for example, fertilized eggs 11 are fixed to the cell fixing plate 2, the cell fixing plate 2 is first stored in the storage chamber 4,
An egg suspension liquid supply tank is placed on the surface of the cell fixing plate 2 while sucking the cell fixing plate 2 from the back side by, for example, 750 mmHg suction pressure through the suction path 44 of the storage chamber 4 by the vacuum pump 5. From 62, the egg suspension is, for example, 0.1 ml / h
Supply at a flow rate of r. This egg suspension contains, for example, about 10 fertilized eggs 11 in 10 ml of a liquid component composed of a culture solution or the like.

In this way, the liquid component of the egg suspension on the surface of the cell-fixing plate 2 tends to flow toward the suction passage 44, but is moved by the ring members 41a and 41b.
The liquid component permeates to the rear surface side through the fine holes 21 in the central portion of the plate 2 because the flow to the peripheral region of the plate is hindered. At this time, since the liquid-impermeable film 31 is formed in a region other than the concave portion 3 of the plate 2, the liquid component cannot pass through the liquid-impermeable film 31, and the liquid component does not pass through the surface of the plate 2. I will be flipped. Therefore, on the surface of the plate 2, the liquid component flows only into the concave portion 3, so that the liquid flow is generated. The fertilized egg 11 contained in the egg suspension is drawn into the recess 3 by riding this liquid flow.

When the fertilized egg 11 is drawn into the concave portion 3, since the concave portion 3 is formed so as to be compatible with the lower surface side of the fertilized egg 11, the fertilized egg 11 is sucked and stored by the entire inner surface of the concave portion 3. When the number of the recesses 3 in which the fertilized eggs 11 are stored increases, the surface of the recesses 3 becomes
And the surface of the region other than the concave portion 3 is impermeable to liquid.
1, the ventilation area is reduced, and the pressure in the storage chamber 4, which was initially about 20 mmHg, gradually decreases. And 25 almost all fertilized eggs 11
Is stored, the pressure in the storage chamber 4 is, for example, 0 to 5 mm.
It rapidly decreases to about Hg.

Therefore, due to this pressure change, the cell fixing plate
Since it is possible to detect that the fertilized eggs 11 have been stored in substantially all of the recesses 3 formed in the storage 2, the pressure in the storage chamber 4 is reduced by reducing the pressure of the vacuum pump 5 to, for example, about 740 mmHg based on this pressure change. The pressure is maintained at about 10 mmHg, and the fertilized egg 11 is fixed to a cell fixing plate by this pressure. The reason why the pressure is reduced in this way is to suppress the deformation of the fertilized egg 11.

In this state, a gene is injected into the fertilized egg 11 by, for example, a microinjection method. On the other hand, the liquid component of the egg suspension liquid sucked by the vacuum pump 5 is temporarily stored in the egg suspension liquid storage tank 51 and circulated and supplied to the egg suspension liquid supply tank 62 through the circulation supply pipe 64.

Here, the permeation speed of the liquid component of the egg suspension through the cell fixing plate 2 increases in proportion to the suction pressure of the vacuum pump 5 according to an experimental example described later. The pressure is preferably about 0.5 to 5 ml / hr. For this purpose, the vacuum pressure of the vacuum pump 5 is, for example, 650 to 760 mm.
Hg is preferred. Plate 2 for cell fixation
The size of the fine holes 21 is preferably, for example, about 2 to 20 μm, and the volume of the fine holes 21 is 0.25 to 2.5 μm.
It is preferably about cc / g, and the thickness of the plate 20 is preferably about 0.5 to 2 mm.

When the fertilized eggs 11 are fixed to the cell fixing plate 2 by the above-described method, a large number of fertilized eggs 11
Can be fixed to a predetermined position of the cell fixing plate 2 in a stable state while suppressing deformation. That is, the recess 3
Is formed so as to be compatible with the lower side of the fertilized egg 11 and the liquid component of the egg suspension is sucked through the fine holes 21 of the porous plate 20, so that it is shown in FIG. As described above, the suction force is dispersed over the entire inner surface of the concave portion 3, and the fertilized egg 11 can be sucked over the entire inner surface. For this reason, since a large suction force is not applied to a part of the fertilized egg 11 at the time of suction, deformation is suppressed.

Further, since the entire lower outer surface of the fertilized egg 11 is suction-fixed to the concave portion 3, the fertilized egg 11 is fixed to the concave portion 3 with a large area, whereby the fixed fertilized egg 11 is stabilized. I do. Therefore, for example, the egg suspension flows into and flows into the fertilized egg 11 already fixed in the concave portion 3 and the fertilized egg 11 in which the liquid component is fixed.
Even if the egg 1 collides, there is no possibility that the fertilized egg 11 fixed from the concave portion 3 jumps out of the concave portion 3 due to the impact force. Furthermore, since it is not necessary to form a suction hole for allowing a liquid to flow through the plate for cell fixation separately, the number of steps for manufacturing the plate for cell fixation is smaller than in the case of forming a suction hole. And the production of the plate becomes easier.

Here, for example, as shown in FIG. 4 (b), a concave portion 32 is formed on one surface of the plate 22, and a suction hole 32a for allowing liquid to flow therethrough is provided at the bottom of the concave portion 32. Assuming that cells are fixed on a plate for use,
Since the egg suspension is sucked through the suction hole 32a, when the fertilized egg 11 is drawn into the concave portion 32, the suction force is concentrated on a part of the fertilized egg 11, and this part is
a, the fertilized egg 11 may be deformed and damaged as shown by a two-dot chain line in the figure, and the stability of the fertilized egg 11 deteriorates because the area fixed by suction is small.

Further, according to the above-described cell fixing method, it is possible to determine whether or not the fertilized eggs 11 have been fixed in all the recesses 3 by detecting the pressure in the storage chamber 4. Can be accurately and easily determined at the end of the operation, and the work efficiency can be improved.

As described above, according to the present embodiment, the fertilized egg 11 can be stably fixed to the cell fixing plate 2, so that the fertilized egg 1 can be fixed by the microinjection method.
Also when inserting the microneedle into 1, the microneedle is easily inserted, and the risk of damaging the fertilized egg 11 at the time of insertion is reduced. The fertilized egg 11 is regularly fixed at a predetermined position on the cell-fixing plate 2, and the upper side of the fertilized egg 11 projects from the cell-fixing plate 2, so that the insertion operation of the microneedle can be easily performed. In other words, the gene can be reliably and easily injected into the fertilized egg 11. Therefore, there is less need to rely on the experience and skill of workers, and work efficiency is improved.
Since the operation of inserting the micro needle is facilitated, the operation time is shortened, and the burden on the operator can be reduced.

The cell fixing plate 2 according to the first aspect of the present invention includes one having a concave portion formed in a shape as shown in FIG. 5, for example. The concave portion 33 shown in FIG. 5 (a) has a shape in which the bottom matches the lower surface of the fertilized egg 11 and
1 is formed so as not to protrude from the surface of the cell fixing plate 2. In such a concave portion 33, an effect similar to that of the above-described cell fixing plate 2 can be obtained. 5B and 5C show the case where the recesses 34 and 35 are formed in the shape of a quadrangular prism or a column, and the example shown in FIG. 5D shows the case where the recess 36 is formed in a conical shape. However, even such a concave portion can be regularly fixed at a predetermined position of the cell fixing plate 2.

In a device used for fixing a fertilized egg to a cell-fixing plate, the storage chamber 4 is not limited to the structure shown in FIG. Good. The storage chamber 4 shown in FIG.
O-rings 45 are provided between the outer peripheral edge of the cell-fixing plate 2 and the rod body 4B and the lower peripheral edge of the cell-fixing plate 2, respectively. ing. In such a storage chamber 4, the O-ring 45 enhances the airtightness between the cell fixing plate 2 and the bottomed cylindrical body 4A and rod body 4B, so that the liquid component of the egg suspension is used for cell fixing. Flowing into the peripheral edge of the plate is suppressed.

Next, an experimental example performed by the present inventors to confirm the effect of the present invention will be described. The experimental apparatus used was the one shown in FIG. 7, but reference numeral 4 in the figure denotes, for example, a cell-fixing plate 2 constructed similarly to the storage chamber 4 shown in FIG.
A simulated egg suspension tank 71 is connected to the upstream side of the storage chamber 4 by a supply pipe 72 in which a roller pump 73 is interposed. The roller pump 73 is arranged in a thermostat. A vacuum pump 75 for sucking the back surface of the cell fixing plate 2 is connected to the suction path 44 of the storage chamber 4 via an exhaust pipe 74. The other end of the exhaust pipe 74 is connected to the vacuum pump 75. A trap 76 is provided. On the upper side of the storage chamber 4, a cell fixing plate 2 is connected via an egg suspension supply port 43.
A camera 77 is provided for observing.

The cell fixing plate 2 has a diameter of 8 m.
m, the thickness is 2 mm, and the diameter of the fine hole 21 is 2 μm.
m and the volume of the micropores 21 were 0.25 cc / g. Recess 3
Had a diameter of 140 μm and a depth of 70 μm. Simulated eggs were prepared by spraying a mixture of a 0.1% aqueous alginic acid solution and a 4% erythrosine aqueous solution on a 1% aqueous calcium chloride solution, and those having a particle size of 155 to 180 μm were selected. A simulated egg suspension was produced by suspending about 25 pieces in 20 ml of pure water.

At a temperature of 37 ° C., the simulated egg suspension tank 71
0.1 ml / hr on the surface side of the cell fixing plate 2
The simulated egg suspension is supplied at a flow rate as described above, and the back side of the cell fixing plate 2 is sucked by the vacuum pump 75 while changing the suction pressure, so that the cell fixing plate 2 of the liquid component of the simulated egg suspension is supplied.
The relationship between the permeation speed and the vacuum pump 75 was measured, and the appearance of the imitation egg being fixed in the concave portion 3 was observed by the camera 77. The permeation rate was determined by measuring the permeation amount of the liquid component of the simulated egg suspension per unit time by solidifying water vapor accumulated in the trap 76 with liquid nitrogen and measuring the weight. The vacuum of the vacuum pump 75 is applied to the storage chamber 4.
The internal pressure was determined by using a mercury pressure gauge and a water column. The result is shown in FIG.

From this experiment, it was confirmed that the liquid component of the simulated egg suspension liquid permeated through the cell-fixing plate to the back surface when suction was performed from the back of the cell-fixing plate 2 at a predetermined pressure. Was. Further, the suction pressure and the permeation speed were in a proportional relationship, and it was recognized that the permeation speed increased as the suction pressure increased, and it was confirmed that the simulated egg was fixed to the recess 3.

Subsequently, the thickness of the cell fixing plate 2 was set to 0.
When the same experiment was carried out at 77 mm, the results shown in FIG. 9 were obtained. It was confirmed that the permeation speed was higher than that when the thickness was 2 mm, and that the simulated egg was fixed to the concave portion 3. In particular, when the drawing pressure was about 700 mmHg, it was confirmed that an appropriate liquid flow was generated on the surface of the cell-fixing plate 2, and the simulated egg was quickly fixed to the recess 3.

[0037]

According to the first aspect of the present invention, a plurality of cells can be fixed at predetermined positions on a cell fixing plate. According to the second to fifth aspects of the present invention, a plurality of cells can be stably fixed at predetermined positions of the cell fixing plate. According to the invention of claim 6, it is easy to determine when the step of fixing the cells to the cell fixing plate is completed.

[Brief description of the drawings]

FIG. 1 is a plan view and a sectional view showing an example of a cell fixing plate according to an embodiment of the present invention.

FIG. 2 is a cross-sectional view showing a main part of a cell fixing plate according to an embodiment of the present invention.

FIG. 3 is a configuration diagram illustrating an example of an apparatus for performing a cell fixing method according to an embodiment of the present invention.

FIG. 4 is a cross-sectional view for explaining the operation of the cell fixing plate.

FIG. 5 is a cross-sectional view showing another example of a cell fixing plate.

FIG. 6 is a sectional view showing another example of the storage room.

FIG. 7 is a configuration diagram showing an experimental apparatus used for an experiment for confirming the effect of the present invention.

FIG. 8 is a characteristic diagram showing a transmission speed and a drawing pressure showing experimental results.

FIG. 9 is a characteristic diagram showing a transmission speed and a drawing pressure showing experimental results.

FIG. 10 is a plan view showing a conventional cell fixing method.

[Explanation of symbols]

 2 Plate for Cell Fixation 20 Porous Plate 21 Micropore 3, 33, 34, 35, 36 Concave Part 31 Non-permeable Membrane 4 Storage Room 43 Egg Suspension Supply Port 44 Suction Path 5 Vacuum Pump

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[Procedure amendment]

[Submission date] February 13, 1997

[Procedure amendment 2]

[Document name to be amended] Drawing

[Correction target item name] All figures

[Correction method] Change

[Correction contents]

FIG.

FIG. 2

FIG. 3

FIG. 5

FIG. 4

FIG. 6

FIG. 7

FIG. 8

FIG. 9

FIG. 10

Continued on the front page (72) Inventor Takayuki Tsuji 5-7 Fujishirodai, Suita-shi, Osaka Inside the National Cardiovascular Center (72) Inventor Toshiya Fujisato 5-7 Fujishirodai, Suita-shi, Osaka National Circulation Inside the disease center (72) Inventor Takeo Nishi 1 Shinmeicho, Yokosuka City, Kanagawa Prefecture Inside the Hibira Toyama Technical Center Co., Ltd. Inventor Fumimasa Yamada 100 Fukuno-cho, Higashi-Tonami-gun, Toyama Pref.

Claims (6)

[Claims] 1. A cell fixing plate, wherein a plurality of recesses for accommodating cells are formed on one surface of a plate made of a liquid-permeable material. 2. The cell fixing plate according to claim 1, wherein the recess is formed in a shape into which a part of the cell fits. 3. The cell-immobilizing plate according to claim 1, wherein a liquid-impermeable membrane is formed in a region other than the concave portion on one surface side of the plate made of a liquid-permeable material. G. 4. The cell fixing plate according to claim 1, wherein the liquid-permeable material is a porous material.
G. 5. The cell fixing plate according to claim 1, wherein the liquid-permeable material is microporous glass. 6. A cell fixing plate in which a plurality of recesses for accommodating cells are formed on one side of a plate made of a liquid-permeable material is accommodated in an accommodation chamber, and cells are contained on one side. In the method of fixing the cells in the concave portion by supplying a liquid and suctioning from the other surface side, detecting a pressure in the storage chamber, and determining whether the cells are fixed in the concave portion based on the detected value. A method for fixing cells, characterized by making a judgment.