JPH0720022A - Method for preparing suspension cell sample - Google Patents

Abstract

PURPOSE:To reduce the destruction of cells and improve the dispersion of the cells by reducing contacts between each cell so that a suspension cell sample can be easily observed in a short time by dipping cellular tissue cut into small pieces in an enzyme solution and collecting cells by centrifugal separation after filtration. CONSTITUTION:In a beaker 1, cell tissue is cut into small pieces with scissors and the pieces are ground down to a desired size. After dipping the pieces in an enzyme solution 5 so that cells can be easily separated from each other, the cells are collected from the solution 5 by filtering the solution 5 through a stainless steel mesh 6, etc., and separating the cells by centrifugal separation. The collected cells are made finer in steps by repeating suction and ejection with a pipet or syringe 11 by an adequate number of times in a suspension liquid 9. Then the suspension liquid 9 is filtered through a nylon mesh, etc., and the filtered liquid 9 is dropped onto a slide glass. After dropping, the liquid 9 is centrifugally smeared by rotating the slide glass.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for preparing a floating cell specimen for cytodiagnosis.

[0002]

2. Description of the Related Art Conventionally, as a method for preparing a floating cell specimen for cytodiagnosis, a desired cell tissue piece is taken out, and this tissue piece is mechanically produced by scissors or a blender, or is produced by an enzyme or a surfactant. There is a method in which the suspension is chemically finely divided, and the suspension containing the finely divided cells is placed on a slide glass and further covered with a cover glass.

[0003]

However, in such a conventional production method, cells are easily broken, cells are easily contacted with each other, and dispersion is poor, so that there is little destruction of cells or contact between cells. It is difficult to always produce a good uniform single-layered sample of the above, and it takes a lot of labor and time to observe the sample with a microscope.

Therefore, the present invention has been made by paying attention to such conventional problems, and reduces the destruction of cells and the contact between cells, and at the same time improves the dispersion of cells, facilitating the observation of specimens and It is an object of the present invention to provide a method for preparing a floating cell specimen that can shorten the observation time.

[0005]

Means for Solving the Problems The method for preparing a floating cell specimen according to the present invention comprises the steps of fragmenting a cell tissue piece, immersing the minced cell tissue in an enzyme solution, filtering the enzyme solution as necessary, and then removing the enzyme. The feature is that cells are collected from the solution by centrifugation, the collected cells are gradually finely divided in the suspension, the suspension is filtered if necessary, and then the suspension is centrifuged on a slide glass. And

More specifically, in the method of the present invention, first, the mechanically finely divided cell tissue is immersed in an enzyme solution to facilitate isolation of cells, and then, if desired, the enzyme solution is treated with a stainless mesh or the like. After filtering, cells are collected from the enzyme solution by centrifugation, and the collected cells are suspended in a suspension and aspirated and ejected (pipetting) with a pipette or aspirated and ejected (syringing) with a syringe, which are repeated a suitable number of times. It is made finer step by step, and subsequently, if necessary, the suspension is filtered using a nylon mesh or the like, the suspension is dropped on a slide glass, and the slide glass is rotated to perform centrifugal smearing.

[0007]

In the method of the present invention, generally, the cells are mechanically finely divided, and then the cells are collected by centrifugation to facilitate biochemical isolation with an enzyme solution, and the collected cells are further finely divided. Since the micronized cells are smeared onto a slide glass by centrifugation, not only the destruction of cells and the contact between cells are reduced, but also the dispersibility of cells on the slide glass is improved, resulting in a uniform monolayer specimen. , The observation of the specimen becomes easy and the observation time is shortened.

[0008]

EXAMPLES The method for preparing a floating cell specimen of the present invention will be described below based on examples. The process drawing is shown in FIGS. First, in FIG. 1, a target cell tissue piece 2a is cut into small pieces with scissors 3 in a beaker 1 having an appropriate size, and further crushed with a glass stopper 4 to obtain a tissue piece 2b having a certain size. (See FIG. 2).

Then, as an enzyme solution, 0.5% collag
Put the enase / Dulbecco's phosphate buffer 5 in the beaker 1 and leave it for about 20 minutes to facilitate isolation of cells of the tissue piece 2b (see FIG. 3). Then, the enzyme solution 5 in the beaker 1 is filtered through the stainless steel mesh 6 having 75 μm holes, and the filtrate 5 ′ is put in another beaker 7 (see FIG. 4). Subsequently, in FIG. 5A, the enzyme solution 5 ′ is transferred to, for example, a test tube 8 having an appropriate size, and the test tube 8 is rotated to perform centrifugation. This causes the cells 2c to settle on the bottom of the test tube 8,
Excess enzyme solution 5'is removed (supernatant removal) to collect cells 2c [see (b) in the figure]. Dulbecc in this cell 2c
Add o's phosphate buffer 9 to resuspend cells 2c [see (c) in the figure].

Next, as shown in FIG. 6, the suspension 9 of the cells 2c is aspirated and ejected at an appropriate speed by a pipette 10,
Repeat this pipetting an appropriate number of times. This allows
The cells 2c in the suspension 9 become finer cells 2d. Succeedingly, as shown in FIG. 7, a syringe 1 provided with a 25 × 1 needle
1, the suspension 9 is sucked and ejected at a rate of 0.4 ml / sec, and this syringe is also repeated an appropriate number of times. As a result, the cells 2d formed by pipetting become finer cells 2e. That is, by pipetting and syringe, the cells 2c collected in the step of FIG. 5 are made finer step by step so as not to damage them.

After syringing, as shown in FIG. 8, 40 μm
The suspension 9 in the test tube 8 is filtered by the nylon mesh 12 having the holes, and the filtrate 9 ′ is put in another test tube 13. By this filtration, the cells 2e in the suspension 9 are isolated, and the isolated cells 2f float in the filtrate 9 '. Then, an appropriate amount of the suspension liquid 9 ′ of the cells 2f is dropped on the slide glass 14,
Rotate this slide glass 14 with a spinner,
By performing centrifugal smearing, a sample of floating cells can be obtained (see FIG. 9).

In this manufacturing method, compared with the conventional method,
It is possible to prepare a uniform monolayer specimen with good dispersion and little cell breakage or contact between cells, and this is shown below by the contact rate (%) and the destruction rate (%). For comparison, the contact rate and the destruction rate by the conventional method in which a sample is prepared mechanically with scissors, a blender or the like or biochemically finely divided with an enzyme, a surfactant or the like are also shown.

[0013] From this, it can be seen that in the present invention, the contact rate and the destruction rate are both considerably lower than in the past, contact between cells is small, cell destruction is small, and cell dispersibility is excellent.

[0014]

As described above, according to the method for preparing a suspension cell specimen of the present invention, the cells are finely divided by a mechanical and biochemical method, and the cells are collected by centrifugation to further make the cells finer. After that, since centrifugal smearing is performed, it is possible to consistently and stably prepare a uniform monolayer specimen with less dispersion of cells and less contact between cells and good dispersion. Therefore, the sample observation by the microscope becomes easy and the observation time becomes short.

[Brief description of drawings]

FIG. 1 is a process chart showing a method of the present invention.

FIG. 2 is a process diagram that follows FIG.

FIG. 3 is a process drawing that follows FIG.

FIG. 4 is a process drawing that follows FIG.

FIG. 5 is a process drawing that follows FIG.

FIG. 6 is a process drawing that follows FIG.

FIG. 7 is a process drawing that follows FIG.

FIG. 8 is a process drawing that follows FIG.

FIG. 9 is a process drawing that follows FIG.

[Explanation of symbols]

 1,7 Beaker 2a to 2f Cell 5 Enzyme solution 6,12 Mesh 8,13 Test tube 9,9 'Suspension liquid 10 Pipette 11 Syringe 14 Slide glass

Claims (1)

[Claims] 1. A cell tissue piece is crushed, the crushed cell tissue is immersed in an enzyme solution, the enzyme solution is filtered if necessary, and then the cells are collected from the enzyme solution by centrifugation, and the collected cells are collected. A method for preparing a suspended cell specimen, which comprises stepwise finely dividing the suspension in a suspension, filtering the suspension as needed, and centrifuging the suspension on a slide glass.