WO2007010924A1

WO2007010924A1 - Gelatinized product of cell, and preparation system for high density cell or tissue array

Info

Publication number: WO2007010924A1
Application number: PCT/JP2006/314237
Authority: WO
Inventors: Jyunya Fukuoka
Original assignee: National University Corporation University Of Toyama
Priority date: 2005-07-21
Filing date: 2006-07-19
Publication date: 2007-01-25
Also published as: JPWO2007010924A1

Abstract

[PROBLEMS] To provide a method for preparation of a cell block from cells dispersed in a medium such as a liquid component of a specimen, for use in the preparation of a cell array; and to provide a system for the preparation of a high dense cell/tissue array. [MEANS FOR SOLVING PROBLEMS] An unused portion of the cells in a specimen submitted for a routine cell-based diagnosis or the like is dispersed in a medium, a mucopolysaccharide is added to the medium to cause the gelation of the medium, the gelated cell material is treated with formalin, and then the formalin-treated gelated cell material is used to prepare a cell block. By using a cell block and/or a tissue block prepared in this manner, it becomes possible to collect/store tissue/cell array samples in many facilities, gather up the samples in a single place, and construct a system for the preparation of highly dense tissue/cell arrays.

Description

Specification

Cell gel tissue and highly integrated cell or tissue array production system

[0001] The present invention relates to a cell gel and a highly integrated cell for producing a cell array.

'Relates to a system for producing tissue arrays.

Background art

[0002] A tissue array is obtained by taking out a small section of tissue force embedded in paraffin and arranging a plurality of pieces on a glass slide.

Currently, devices using the method developed by Kononen et al. (Non-patent Document 1) are commercially available.

In addition, a method improved by the method of Kononen et al. Has been reported (Non-Patent Document 2, Patent Document 1, Patent Document 2).

In the tissue array, usually (1) the target tissue is taken out from the donor block of the paraffin-embedded tissue in a cylinder of several millimeters with a cylinder or the like.

(2) Move the hole to receive the extracted cylindrical tissue to the recipient block that has been placed in advance.

(3) The recipient block is made into thin slices and placed on a slide glass (Patent Document 1).

On the other hand, for cell arrays using cells instead of yarn and weave, for example, a method of producing force by suspending frozen cells by collecting cells collected from a surgical procedure or biopsy sample is known. (Patent Document 1).

[0003] Non-patent literature l: Nature Medicine, 4 (7), 844—847 (1998)

Non-patent document 2: Medical examination, 52 (6), 806-811 (2003)

Patent Document 1: Special Table 2004-500891

Patent Document 2: JP 2004-258017 A

Disclosure of the invention

Problems to be solved by the invention [0004] With the tissue force embedded in norafin or the like, a technique for producing a tissue array is almost established!

However, as a technique for producing a cell array from the surplus of specimens submitted for cytodiagnosis or the like, for example, although there is a method of using cells suspended in a frozen embedding material described in Patent Document 1, there is a method for producing a microarray. It is necessary to add a cooling device or the like to the apparatus.

In addition, to collect a considerable number of uniform disease groups, it is necessary to use samples collected at many facilities, but it is not reasonable to install special equipment at many facilities.

In addition, frozen specimens have low storability of morphology and are suitable for observing minute structures under a microscope. Tissue arrays and cell arrays are indispensable as research and treatment / diagnosis / prognostic determinants. Their production requires special machines and techniques.

On the other hand, in order to collect a considerable number of uniform disease groups, it is necessary to use samples collected at many facilities.

From this background, it was actually difficult to produce hundreds and thousands of tissue / cell arrays.

Means for solving the problem

[0005] In order to prepare a cell array, the present inventor disperses surplus cells of a specimen submitted for daily cytodiagnosis and the like in a medium, and then adds mucopolysaccharide to the dispersion to obtain a gel gel. After that, a cell block was prepared using a formalin-treated cell gel, and a method for preparing a cell array from this cell block was devised.

Furthermore, a method for efficiently and temporarily storing specimens collected at a large number of facilities, a sorting container used for the method, and a cell 'tissue specimen temporarily stored in the container is efficiently collected, and the degree of accumulation is high. A system for creating cell and tissue arrays was devised.

Hereinafter, the present invention will be described in detail.

[0006] Examples of the medium for dispersing cells include a liquid component of the specimen itself, a physiological saline, a phosphate buffered physiological saline, and the like.

Examples of the mucopolysaccharide include hyaluronic acid, chondroitin, chondroitin sulfate, and alkali metal salts thereof.

Preferred is sodium hyaluronate. The amount of mucopolysaccharide added is sufficient if the cell suspension is gelled, for example, 5 to 20 times the amount of cell suspension (WZV).

Formalin treatment may be performed, for example, using neutral buffered formalin 2-10 times the amount of gel (WZW) at room temperature for 6-12 hours.

During this time, it is better to shake the container at a low speed.

[0007] The cell gel and the cell block of the present invention can be produced, for example, by the following method.

(1) The remaining cells used for diagnosis among the cells collected for cytodiagnosis are centrifuged (200-3000 rpm for 3-5 minutes) to form a cell mass (pellet). Discard the supernatant leaving the same amount of liquid component.

If the cell mass is small, an appropriate amount of supernatant is left.

(2) Stir approximately the same amount of the remaining liquid component as the pellet and distribute the cells uniformly in the liquid. Here, a dye (such as matoxylin for dilution) may be added to make the gel easily visible.

(3) Collect a certain amount of the cell suspension, transfer it to a container, add 5-20 times (WZ V) mucopolysaccharide of the cell suspension, and stir further.

(4) Leave at around 4 ° C for 1-2 hours.

(5) After confirming that the gel has solidified, use 2-10 times the amount of static buffer (WZW) neutral buffered formalin! ]

(6) Leave at room temperature.

(7) Scrape out the obtained cell block with a medicinal spoon, etc., and put it in the process of creating a normal tissue paraffin block.

Moreover, paraffin embedding is performed manually by a normal method.

[0008] Next, a method for collecting samples for a multicenter force cell array and Z or tissue array and producing a cell array and z or tissue array having a high sample force will be described.

(&) 11 columns 111 columns 1 to 5111111 diameter 1-10 mm deep perforated paraffin block (3cm in length 4cm in height lcm can be used or larger) I mean.

Here, n and m mean an integer of 3 to 10.

Preferred is n = 5, m = 5, 25 in total, 2mm in diameter and 5mm in depth

(b) A hollow needle with an inner diameter of 1 to 5 mm for sample collection (a biopsy needle is acceptable) and a paraffin block (a) are distributed to many hospitals.

(c) At the same time, a container that can be stored in 5 rows x 5 rows or 10 rows x 10 rows is handed over as a container for temporarily storing the paraffin block of (a).

This container is divided to a size that fits a norafin block and consists of 5 rows x 5 rows or 10 rows x 10 rows!

For example, in the case of a paraffin block using a normal mold, each partition is 3.2 cm x 4.5 cm large, and the overall size of the box is approximately 16 cm x 22.5 cm x 3.5 cm or 32 cm x 45 cm x 3 It is 5cm.

(d) When a specimen comes out in daily work, remove the part with a hollow needle.

(It is preferable to use one having a mechanism for pushing out the contents of the hollow needle.)

(d) Distribute the extracted cores in advance and embed them in the n-row x m-row holes of the paraffin block of (a).

(e) Collect the paraffin block (first block) when the core embedding reaches the planned number. Collecting can be done in block units or temporary storage containers!

(f) The tissue / cell embedded in the recovered paraffin block is transplanted to about 1000 tissue 'cell array blocks (second block) by an arrayer, for example, Beecher TM Arrayer.

(g) —If three cores, for example, 0.6 mm cores from 2 mm cores are sampled, three blocks with the same conditions can be made at the same time.

(h) —Each block force can produce about 300 usable sections.

When the three blocks are combined, a good quality tissue of about 900 to 1000 sheets. Cell array slides can be sliced.

(i) When slicing, transfer with tape. At the time of slicing, apply tape one by one and slice it to a thickness of 4-5 / ζ πι.

(j) Float a piece of tape on the water, pick it up with a glass slide glass that has been bonded (normal silane coating or MAS coating), and push out moisture and bubbles.

(k) Apply the tissue and slide firmly by drying for more than 2 weeks.

(1) Do not remove the tape until use. (This protects the organization)

(m) When dyeing, the tape is peeled off in xylene, and the dyeing process is started as in the case of ordinary paraffin chips.

The invention's effect

[0010] According to the first aspect of the present invention, a cell paraffin block can be created even if sufficient cells are not obtained from the surplus specimens of specimens submitted for daily cytodiagnosis. In addition, according to the second aspect of the present invention, it is possible to easily construct a system for producing an array by storing tissue 'collecting a sample for a cell array' at a large number of facilities and collecting them in one place. I'll do it.

Brief Description of Drawings

[0011] FIG. 1 is a photograph of a donor block.

[Fig.2] Photograph of stained cell / tissue array.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, embodiments of the present invention will be described with reference to examples and test examples, but the present invention is not limited to these.

Example 1

From the cells collected for cytodiagnosis, the remaining cells used for diagnosis are centrifuged (3000 rpm, 5 minutes) to obtain a cell mass (pellet).

Discard the supernatant leaving approximately the same amount of liquid component as the cell mass.

Next, stir approximately the same amount of the remaining liquid component with the cell mass to distribute the cells uniformly in the liquid (if the cell mass is small, leave 200 μL of the supernatant).

Collect 200 L of cell suspension and transfer to a 1.5 mL tube.

Add 5 mg of sodium hyaluronate, stir it, and leave it at 4 ° C for 1 hour. After confirming that the cell suspension has solidified in a gel, gently add 1 mL of neutral buffered formalin. And leave at room temperature.

Squeeze the fixed gel mass with a medicinal spoon and embed it in a paraffin block using the same procedure as for normal pathological tissue.

Then, remove the core with a hollow needle and transfer it to a pre-prepared paraffin block (2 mm diameter, 5 mm deep holes arranged in 5 rows x 5 rows, 30 mm long x 40 mm wide x 10 mm high: one donor block) .

[0013] Example 2

Samples for the array were prepared simply by the following procedure.

1. Use a needle with a diameter of 2 mm in a normal paraffin block that does not contain tissue, and drill a total of 25 holes of 5 mm in length and 5 mm in depth, 5 in total (donor block). (photo1)

2. Distribute 2mm diameter hollow needle or 2mm core biopsy needle and donor block to other facilities.

At the same time, we will distribute 5 x 5 partitions (16 cm x 22.5 cm x 3.5 cm) with a size that fits the donor block exactly (each is 3, 2 cm x 4.5 cm).

3. In the facility where the donor block is distributed, if cells or tissue specimens that match the research purpose are obtained from cytology or excised tissue, etc., the cell block is prepared by the method of Example 1 or the like, or Remove the necessary part from the tissue block itself with a hollow needle with a diameter of 2 mm or a biopsy needle, and put the contents into the hole of the donor block.

4. From the donor block filled with the sample and sent back to the other facility, the sample is removed from the donor block by using an array production device, for example, Beecher TM Arrayer (manufactured by Beecher Instruments). Transplanted to the recipient block).

[0014] The following procedure is performed.

(1) Take three 0.6 mm cores from one core (2 mm) of the donor block and transplant each to three recipient blocks with 990 0.6 mm cores.

(2) Make a slide slice from the recipient block with a microtome.

When slicing, apply tape one by one and slic it to a thickness of 4-5 / ζ πι. (3) Float the tape with the slices in water and pick it up with an adhesive-processed slide glass (a normal sill coat or MAS coat may be used) to push out moisture and bubbles.

(4) Let it dry for more than 2 weeks.

In order to protect the tissue after drying, the tape is not removed until dyeing or the like.

(5) When dyeing, remove the tape in xylene and dye as it is for normal paraffin sections.

Industrial application fields

Protein profiles for each disease (especially cancer) can be created, and these clusters will elucidate the factors that determine treatment, diagnosis, and prognosis, and contribute to new treatment selection 'disease classification'.

It is difficult for tissues to compare the lesion before treatment and the lesion after treatment, but in the case of cells, it is often easy to secure a specimen.

It can be applied to judge resistance to drugs, elucidate the mechanism, or to judge secondary drug selection.

Claims

The scope of the claims

[1] A cell gel product obtained by adding mucopolysaccharide to a cell dispersion and gelling it, followed by formalin treatment.

[2] The cell gel product according to claim 1, wherein the mucopolysaccharide is hyaluronic acid.

[3] A cell block obtained by collecting a sample from the cell gel product according to claim 1 or 2, and embedding the collected sample in a baraffin.

[4] Distribute the following first block (A) and container (B) to the facility where the system is constructed in advance, and use the container (B) at the distributed facility to temporarily store the first block (A). After the first block (A) reaches the predetermined number, after the container (B) is recovered, the recovered first block (A) force is created, and then a plurality of second blocks are produced. A highly integrated cell or tissue array production system characterized by producing a cell or tissue array.

(A): A first block containing the cell block and Z or tissue specimen according to claim 3.

(B): A container for storing (A) in an aligned manner.

[5] The first block consists of n rows x m rows (n and m are integers from 3 to 10), diameter l to 5mm, depth 1 to

5. The highly integrated cell or tissue array production system according to claim 4, wherein the system is a paraffin block having 10 mm holes.

[6] The highly integrated cell or tissue array production system according to claim 4, wherein the container (B) is a container partitioned by 5 rows x 5 rows or 10 rows x 10 rows .